<p class="jove_content" style=";text-align:right;direction:rtl"> עבודה זו נתמכה על ידי תומאס פ וקייט מילר Jeffress Memoria Trust (עד דצמבר) ו- NIH להעניק R35GM119617 (עד דצמבר). הדמית מיקרוסקופ confocal בוצעה לעבר Core אפיון ננו VCU (NCC) המתקן. </p>

<p class="jove_title" style=";text-align:right;direction:rtl"> 1. השג Nesprin-2G חיישן דנ&quot;א האחר פלסמיד דנ&quot;א </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השג Nesprin-2G TS (חיישן המתח), Nesprin-2G HL (ללא ראש) מלאה, mTFP1, ונוס, ו TSmod ממקור מסחרי. הפץ כל פלסמידים DNA ולטהר אותם באמצעות זני <em>חיידק</em> סטנדרטיים, כגון-α DH5, כפי שתואר לעיל <sup class="xref"><sup>12,</sup></sup> <sup class="xref">13.</sup> </li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 2. תאי Transfect עם Nesprin-2G ו- DNA פלסמיד אחר </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדל תאי פיברובלסטים NIH 3T3 כדי מפגש 70-90% בצלחת תרבית תאי 6-היטב בתוך חממת תרבית תאים סטנדרטית עם טמפרטורה (37 מעלות צלזיוס) ו <sub>2</sub> CO (5%) תקנה. עבור מדיום צמיחת תאים, השתמש בינוני נשר Modified של Dulbecco (DMEM) בתוספת 10% בסרום עגל עובר. </li><li style=";text-align:right;direction:rtl"> במנדף תרבית תאים, להסיר את המדיום ולשטוף היטב עם כל כ 1 מ&quot;ל של מדיום התא מופחת-סרום. הוספת 800 μL של המדיום הסלולרי מופחת-סרום היטב כלומקום בתא 6-היטב החממה. </li><li style=";text-align:right;direction:rtl"> פיפטה 700 μL של המדיום הסלולרי מופחת-סרום לתוך צינור 1.5 מ&quot;ל עם 35 μL של פתרון המוביל השומנים כדי ליצור את &quot;lipomix&quot;. מערבבים ידי pipetting. סמן הצינור עם &quot;ל&quot; </li><li style=";text-align:right;direction:rtl"> אסוף שישה 1.5 צינורות מ&quot;ל ו לתייג אותם 1 עד 6. פיפטה 100 μL של המדיום הסלולרי מופחת-סרום לתוך צינור אחד. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות ריכוז פלסמיד דנ&quot;א משלב 1, פיפטה 2 מיקרוגרם של Nesprin 2G-TS לתוך צינורות 1 ו 2. פיפטה 2 מיקרוגרם של Nesprin-HL לתוך צינורות 3 ו 4. פיפטה 1 מיקרוגרם של mTFP לתוך צינור 5. פיפטה 1 מיקרוגרם של mVenus לתוך הצינור 6. אל תשתמש שוב טיפים פיפטה כאשר pipetting סוגים שונים של דנ&quot;א. </li></ol></li><li style=";text-align:right;direction:rtl"> פיפטה 100 μL של lipomix מהצינור &quot;L&quot; לתוך צינור אחד שכותרתו (1-6) ומערבבים על ידי pipetting חזר. השתמש פיפטה נקייה עבור כל צינור. דגירה של 10-20 דקות. </li><li style=";text-align:right;direction:rtl"> הוספת 200 μL מצינור כל שכותרתו באר בתא 6-היטב עם 70-90% ומחוברותתאים. סמן בראש כל טוב עם DNA המקביל הוסיף. מניח את חדר 6-היטב באינקובטור במשך 4-6 שעות. </li><li style=";text-align:right;direction:rtl"> לשאוב בינונית ומוסיפים 1-2 מ&quot;ל של המדיום הסלולרי מופחת-סרום כדי לשטוף. לשאוב את המדיום הסלולרי-סרום מופחת, להוסיף 2 מ&quot;ל של טריפסין לכל היטב, ומניחים בצלחת 6-היטב החממה (5-15 דקות). </li><li style=";text-align:right;direction:rtl"> בעוד תאים לנתק בחממה, זכוכית התחתונה מעיל 6 לצפייה מנות עם שכבה של פיברונקטין בריכוז של 20 מיקרוגרם / מ&quot;ל ​​מומס מלוחים פוספט שנאגרו (PBS). אפשר את הכלים כדי מעיל השטח בשכונה תרבית תאים (כ 20 דק &#39;). </li><li style=";text-align:right;direction:rtl"> לנטרל את טריפסין על ידי הוספת 2 מ&quot;ל של DMEM פעם התאים מנותקים. </li><li style=";text-align:right;direction:rtl"> מעבירים את תכולת כל מהבאר אל צינור צנטריפוגה 15 מ&quot;ל שכותרתו ומסובב ב 90 XG במשך 5 דקות בצנטריפוגה הרוטור מתנדנד. לשאוב supernatant מחדש להשעות כל גלולה תא 1000 μL של DMEM ידי ערבוב פיפטה. </li><li style=";text-align:right;direction:rtl"> לשאוב את תערובת פיברונקטין מןמנות זכוכית פיפטה 1000 μL של כל השעית תא על צלחות הזכוכית. </li><li style=";text-align:right;direction:rtl"> אחרי התאים ליישב לתחתית של מנות זכוכית (~ 15 דקות), להוסיף 1 מ&quot;ל של DMEM + 10% FBS + 1% פן-סטרפ היטב כל מקום בחממה התא. אפשר התאים לצרף ולהביע חיישן לפחות 18-24 h. <br /> הערה: תאים הם transfected באמצעות ריאגנטים transfection השומנים מסחרי קטיוני (ראו <strong>טבלה של חומרים).</strong> לחלופין, שורות תאים יציבות ניתן לבחור באמצעות פלסמיד עם גן המקנה עמידות רעלן (פלסמידים pcDNA עבור Nesprin-TS ו -HL זמינים באתר מאגר DNA (ראה <strong>טבלה של חומרים)</strong> לספק תאים המבטאים התנגדות geneticin ). בנוסף, שיטות זיהום ויראלי (lentivirus, רטרו-וירוס, או אדנו) יכול להיות מנוצל כדי להביע את החיישן בתאים שקשה transfect. </li><li style=";text-align:right;direction:rtl"> בנוסף Nesprin-TS, transfect תאים נוספים עם Nesprin HL אפס עבורמלא CE, כמתואר בשלבי 2.1-2.12; TSmod יכול לשמש גם כשלט אפס כוח צריך להפגין סריג דומה Nesprin-HL. </li><li style=";text-align:right;direction:rtl"> תאים Transfect עם mTFP1 ו ונוס ליצור טביעות אצבעות ספקטרלי (ראה שלב 4). <br /> הערה: mTFP1 ו ונוס בדרך כלל לבטא ברמות גבוהות יותר מאשר Nesprin-TS, וככזה, כמויות נמוכות של DNA ייתכן שיהיה צורך טרנספקציה להשיג רמות ביטוי דומה. </li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 3. בדוק יעילות transfection </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בערך 18-24 שעות לאחר השלמת transfection, להשתמש מיקרוסקופ פלואורסצנטי הפוכה כדי לבחון את היעילות של transfection ידי השוואת מספר התאים פלורסנט למספר הכולל של תאים לאור (בדרך כלל 5-30%). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש מיקרוסקופ פלואורסצנטי מצויד תדר עירור ליד 462 ננומטר (mTFP1) או 525 ננומטר (ונוס) ו מסנן פליטה מרוכז ליד 492 ננומטר (mTFP1) או 525 ננומטר (ונוס). <br /> הערה: לחלופין, מערכות סינון GFP תהיה ללכוד שילוב של mTFP1 ו veפליטת Nus ותאפשר את האישור של יעילות transfection. </li></ol></li><li style=";text-align:right;direction:rtl"> תאי תמונת חיה בתוך 48 שעות לאחר transfection. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחלופין, לתקן תאים paraformaldehyde 4% (ב PBS עם סידן ומגנזיום) עבור 5 דקות, חנות PBS, ולהציג לאחר 48 h <sup class="xref">8.</sup> <br /> הערה: Beyond 48 h, איכות האות וכוח דועך. תיקון התאים משמר המדינה שלהם, כולל סריג לידי ביטוי <sup class="xref">8;</sup> עם זאת, תאים צריכים להיות צלמו רק ב- PBS, כמו הרכבה בינונית עשויה להשפיע <sup class="xref">14</sup> סריג. תאים קבועים ניתן להשוות רק לתאים קבועים אחרים, כמו ייתכנו שינוי הביטוי. <br /> זהירות: Paraformaldehyde רעיל. ללבוש ציוד מגן אישי מתאים (PPE). </li></ol></li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 4. לכידת ספקטרלי טביעות אצבעותיהם של mTFP1 ו ונוס fluorophores עבור ספקטרלי unmixing </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במנדף תרבית תאים, להחליף את המדיום הסלולרי עם mediu הדמיהמ &#39;(HEPES שנאגר) השלים עם 10% בסרום שור עובר. </li><li style=";text-align:right;direction:rtl"> מניחים את הכלים הצפייה באופן מבוקר טמפרטורה (37 מעלות צלזיוס) בשלב מיקרוסקופ confocal. </li><li style=";text-align:right;direction:rtl"> מניח את צלחת הצצה מהזכוכית עם תאים-טרנספקציה mTFP1 מעל המטרה השמנה בהגדלה 60X עם צמצם מספרי של 1.4. <br /> הערה: שמן משמש על מיקרוסקופ סורק לייזר על המטרה 60X להידמות מקדם השבירה מקרוב של מצע זכוכית. השמן הוא מונח על גבי העדשה האובייקטיבית שבא במגע ישיר עם coverslip הזכוכית. </li><li style=";text-align:right;direction:rtl"> אתר mTFP1 לבטא בתאים עם מקור עירור 458 ננומטר ו מסנן bandpass פליטה המרוכז ב 500 ננומטר. </li><li style=";text-align:right;direction:rtl"> עם תא פלורסנט בתחום הראייה, לבחור את מצב זיהוי ספקטרלי ( &quot;מצב למבדא&quot; בתוכנה המשמשת כאן) ו ללכוד את התמונה ספקטרלי <strong>(איור 3-1);</strong> כל החדרים כוללים תדרים מעבר 458 ננומטר באמצעות 10 במרווחים ננומטר <strong>(איור 3-3).</strong> בחר fluoresc בהירבאזור אף אוזן גרון (ROI) על התא (רדיוס 20-פיקסל). <br /> הערה: צורת הספקטרלית של ROI mTFP1 להביע צריכה להישאר קבועה יחסית על פני התא. אם הצורה משתנית במידה ניכרת, מחדש ולהתאים את הליזר ולהשיג הגדרות כדי לשפר את יחס אות לרעש. </li><li style=";text-align:right;direction:rtl"> מוסיפים את ההחזר על ההשקעה פלורסנט אומר, עוצמת מנורמל למסד הנתונים ספקטרלי על ידי לחיצה על &quot;שמור ספקטרום למסד הנתונים.&quot; </li><li style=";text-align:right;direction:rtl"> ייעל את כוח הליזר ולהשיג כזה כי מושגת יחס אות לרעש טוב. הגדרות תשתנינה עבור ציוד שונה. השימוש הלא-ניאון, תאים untransfected כהפניה רקע, להגדיל את הרווח ואת הכוח עד תאים בהירים, אך לא מעבר לטווח הדינמי של גלאי (נקודת הרוויה). תאי רקע לא צריכים קרינה לזיהוי לאחר החישוב ממוצע. </li><li style=";text-align:right;direction:rtl"> חזור על התהליך עבור תאי ונוס-טרנספקציה עם החריגים הבאים: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ודא כי תדירות העירור היא 515 ננומטר וכי מסנן bandpass הוא CEntered ליד 530 ננומטר כאשר איתור תאי-להביע ונוס. </li><li style=";text-align:right;direction:rtl"> &quot;מצב ספקטרלית,&quot; להשתמש בתדירות עירור של 515 ננומטר במקום 458 ננומטר. </li></ol></li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 5. לכידת תמונות צרופות </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לעבור את המצב הלכיד &quot;ספקטרלי unmixing.&quot; </li><li style=";text-align:right;direction:rtl"> מוסיפים את טביעות האצבעות ספקטרלי של ונוס mTFP1 לתוך ערוצי unmixing <strong>(איור 3,</strong> חץ-2). </li><li style=";text-align:right;direction:rtl"> הגדר את הלייזר עירור בחזרה למקור ארגון 458 ננומטר. </li><li style=";text-align:right;direction:rtl"> מניחים את צלחת לצפייה Nesprin-TS מעל המטרה שמן 60X. </li><li style=";text-align:right;direction:rtl"> לאחר התמקדות תא פלורסנט עם ביטוי בהיר מספיק, להתאים את הכוח רווח לייזר כדי לייעל את יחס אות לרעש. <br /> הערה: מאז הסריג היעיל של Nesprin-TS הוא ליד 20%, התמונה ונוס הצרופה צריכה להיות דימר משמעותי יותר מתדמית mTFP1 הצרופה. לאחר הגדרת כוח ורווח מקובל נקבעה באופן איטרטיבי, פרמטרים אלה חייבים להישאר קבועים עבור כל סרן התמונותured. </li><li style=";text-align:right;direction:rtl"> לכידת מינימום של 15-20 תמונות של תאי Nesprin-TS עם בהירות דומה יחסית ולהימנע הרוויה פיקסל מוגזמת (כל פיקסלים הרוויים מוסרים במהלך עיבוד תמונה). </li><li style=";text-align:right;direction:rtl"> חזור על התהליך לכיד תמונה עם תאי Nesprin-HL באמצעות פרמטרי הדמיה זהים. </li></ol><p class="jove_title" style=";text-align:right;direction:rtl"> 6. עיבוד תמונה וניתוח תמונה רציו </p><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש בתוכנות קוד פתוח ImageJ (פיג&#39;י) (http://fiji.sc/), תמונות בפורמט טבעיות פתוחות באמצעות קורא BioFormats. </li><li style=";text-align:right;direction:rtl"> בתהליך קדם התמונות ולחשב את התמונות יחס באמצעות פרוטוקולים הוקמו בעבר <sup class="xref">15.</sup> </li></ol>

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<p class='jove_content'>The authors have nothing to disclose.</p>

<p class="jove_content" style=";text-align:right;direction:rtl"> שיטה והדגמה של הדמיה תא חי של מתח מכני על פני Nesprin-2G, חלבון במתחם LINC גרעיני, היה שתוארו לעיל. לפני עבודה זו, טכניקות שונות, כגון שאיפת micropipette, מגנט חרוז cytometry, ליזר-אבלציה מיקרוסקופי, שמשו ליישם עומס על גרעין התא כדי למדוד תכונות החומר בתפזורת שלה <sup class="xref"><sup>16,</sup></sup> <sup c...

<p class="jove_content" style=";text-align:right;direction:rtl"> חיל-רגיש, חיישני סריג מקודדים גנטיים צמחו לאחרונה ככלי חשוב למדידת כוחות מבוסס מתיחה בתאים חיים, ומספקים תובנות איך מכני כוחות מוחלים על פני חלבוני <sup class="xref"><sup>1,</sup></sup> <sup class="xref"><sup>2,</sup></sup> <sup class="xref"><sup>3,</sup></sup> <sup class="xref">4.</sup> באמצעות כלים אלה, חוקרים יכולים דימוי הלא פולשני כוחות תאיים בתאים חיים באמצעות מיקרוסקופי פלורסנט קונבנציונליים. חיישנים אלה מורכבים-זוג סריג (פלורסנט תורם acceptor חלבונים, בתדירות הגבוהה ביותר תורם כחול acceptor הצהוב) המופרדים על ידי פפטיד אלסטי <sup class="xref">3.</sup> בניגוד C- או N-terminal תיוג, חיישן זה מוכנס לתוך אתר פנימי של חלבון כדי למדוד את הכח המכאני ישודר החלבון, מתנהג כמו מד לחץ מולקולרי. גברת מתח מכאני על פני תוצאות החיישן בעל טווח מוגדל בין-p הסריגאוויר, דבר שגרם לפגיעה סריג <sup class="xref">3.</sup> כתוצאה מכך, הסריג הוא ביחס הפכו כוח מתיחה. </p><p class="jove_content" style=";text-align:right;direction:rtl"> חיישנים פלורסנט מבוסס אלו פותחו עבור חלבונים דבקים מוקדים (vinculin <sup class="xref">3</sup> ו תלין <sup class="xref">4),</sup> חלבוני cytoskeletal (α-actinin <sup class="xref">5),</sup> וחלבוני צומת תא-תא (E-cadherin <sup class="xref"><sup>6,</sup></sup> <sup class="xref">7,</sup> VE-cadherin <sup class="xref">8,</sup> ו PECAM <sup class="xref">8).</sup> הנפוץ ביותר בשימוש היטב מאופיין ומקשר אלסטי ב חיישנים אלה נקרא TSmod והוא מורכב מרצף חוזר ונשנה של 40 חומצות אמינו, (GPGGA) <sub>8,</sub> אשר נגזרו flagelliform חלבון משי עכביש. TSmod הוכח להתנהג כמו היענות ננו-אביב אלסטי ליניארי, עם סריג 1 עד 5 pN בכוח מתיחה <sup class="xref">3.</sup> באורכים שונים של flagelliform יכולים לשמש כדי לשנות את r הדינמיתAnge של TSmod סריג כוח רגישות <sup class="xref">9.</sup> בנוסף flagelliform, spectrin חוזרת <sup class="xref">5</sup> ו פפטיד כיסוי ראש villin (המכונה HP35) <sup class="xref">4</sup> שימשו כמו פפטידים אלסטי בין-זוגות סריג ב חיישנים כוח דומה <sup class="xref">4.</sup> לבסוף, דו&quot;ח אחרון הראה כי TSmod יכול לשמש גם כדי לזהות כוחות דחיסה <sup class="xref">10.</sup> </p><p class="jove_content" style=";text-align:right;direction:rtl"> אנחנו לאחרונה פיתחה חיישן כוח עבור והמקשר של-שלד התא Nucleo (LINC) חלבון מורכב Nesprin2G באמצעות TSmod מוכנס לתוך חלבון שפותחו בעבר קטועה Nesprin2G המכונה מיני-Nesprin2G <strong>(איור 2 ג),</strong> אשר מתנהג באופן דומה אנדוגניים Nesprin-2G <sup class="xref">11.</sup> קומפלקס LINC מכיל חלבונים מרובים מפנים מבחוץ לחלק הפנימי של הגרעין, המקשר את שלד תא ציטופלסמית אל למינה הגרעיני. Nesprin-2G הוא חלבון מבני מחייב הוא אתcytoskeleton אקטין בציטופלסמה וכדי חלבונים SUN במרחב perinuclear. שימוש biosensor שלנו, הצלחנו להראות כי Nesprin-2G כפוף מתח actomyosin התלוי NIH3T3 פיברובלסטים <sup class="xref">2.</sup> זו הייתה הפעם הראשונה בכח כי נמדד ישירות דרך חלבון במתחם LINC גרעיני, וזה עשוי להיות כלי חשוב להבין את התפקיד של כוח על הגרעין ב mechanobiology. </p><p class="jove_content" style=";text-align:right;direction:rtl"> פרוטוקול להלן מספק מתודולוגיה מפורט של אופן השימוש בחיישן כוח Nesprin-2G, כולל את הביטוי של חיישן המתח Nesprin (Nesprin-TS) בתאי יונקים, כמו גם רכישת וניתוח של תמונות סריג של תאים המבטאים Nesprin- TS. באמצעות מיקרוסקופ confocal הפוך מצויד בגלאי ספקטרלי, תיאור של איך למדוד פליטה רגישה סריג באמצעות unmixing רפאי הדמית סריג ratiometric מסופקת. תמונות ratiometric הפלט ניתן להשתמש כדי להפוך קואה יחסיתהשוואות כוח ntitative. בעוד פרוטוקול זה מתמקד בביטוי של Nesprin-TS ב פיברובלסטים, זה ניתנת להתאמה בקלות לתאי יונקים אחרים, כולל שתי שורות תאים תאים ראשוניים. יתר על כן, פרוטוקול זה בכל קשור לרכישת תמונה וניתוח סריג ניתן להתאים בקלות חיישני כוח סריג מבוסס אחרים שפותחו עבור חלבונים אחרים. </p>

<table><tbody><tr><td>Nesprin-TS DNA</td><td>Addgene</td><td>68127</td><td>Retrieve from https://www.addgene.org/68127/</td></tr><tr><td>Nesprin-HL DNA</td><td>Addgene</td><td>68128</td><td>Retrieve from https://www.addgene.org/68128/</td></tr><tr><td>mTFP1 DNA</td><td>Addgene</td><td>54613</td><td>Retrieve from https://www.addgene.org/54613/</td></tr><tr><td>mVenus DNA</td><td>Addgene</td><td>27793</td><td>Retrieve from https://www.addgene.org/27793/</td></tr><tr><td>TSmod DNA</td><td>Addgene</td><td>26021</td><td>Retrieve from https://www.addgene.org/26021/</td></tr><tr><td>Competent Cells</td><td>Bioline</td><td>BIO-85026</td><td></td></tr><tr><td>Liquid LB Media</td><td>ThermoFisher</td><td>10855001</td><td>https://www.thermofisher.com/order/catalog/product/10855001</td></tr><tr><td>Solid LB Bacterial Culture Plates</td><td>Sigma-Aldrich</td><td>L5667</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&amp;amp;region=US</td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>273 BR 07335</td><td>SmartSpec Plus</td></tr><tr><td>quartz cuvette</td><td>Biorad</td><td>1702504</td><td>Cuvette for SmartSpec Plus</td></tr><tr><td>DNA isolation kit</td><td>Macherey-Nagel</td><td>740412.5</td><td>NucleoBond Xtra Midi Plus</td></tr><tr><td>6-well cell culture dish</td><td>Falcon-Corning</td><td>353046</td><td>Multiwell 6-well Polystrene Culture Dish</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium, (DMEM) cell media</td><td>Gibco</td><td>11995-065</td><td>DMEM(1x)</td></tr><tr><td>Bovine Serum</td><td>Life Technologies</td><td>16170-078</td><td></td></tr><tr><td>reduced serum cell media</td><td>Gibco</td><td>31985-070</td><td>Reduced Serum Medium, &quot;optimem&quot;</td></tr><tr><td>Lipid Carrier Solution</td><td>invitrogen</td><td>11668-019</td><td>Lipid Reagent, &quot;Lipofectamine 2000&quot;</td></tr><tr><td>1.5 mL sterile plastic tube</td><td>Denville</td><td>c2170</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>0.25% Trypsin-EDTA (1x)</td></tr><tr><td>glass-bottom microscope viewing dish</td><td>In Vitro Scientific</td><td>D35-20-1.5-N</td><td>35 mm Dish with 20 mm Bottom Well #1.5 glass</td></tr><tr><td>Fibronectin</td><td>ThermoFisher</td><td>33016015</td><td>fibronectin human protein, plasma</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190-144</td><td>Dulbecco&#039;s Phosphate Buffered Saline</td></tr><tr><td>15 mL sterile centrifuge tube</td><td>Greiner bio-one</td><td>188261</td><td></td></tr><tr><td>swinging rotor centrifuge&amp;nbsp;</td><td>Thermo electron</td><td>Centra CL2</td><td>Swinging rotor thermo electron 236</td></tr><tr><td>cell culture biosafety hood</td><td>Forma Scientific</td><td>1284</td><td></td></tr><tr><td>climate controlled cell culture incubator</td><td>ThermoFisher</td><td>3596</td><td></td></tr><tr><td>inverted LED widefield fluorescent microscope</td><td>Life technologies</td><td>EVOS FL</td><td></td></tr><tr><td>Clear HEPES buffered imaging media</td><td>Molecular Probes</td><td>A14291DJ</td><td></td></tr><tr><td>Fetal bovine Serum</td><td>Life technologies</td><td>10437-028</td><td></td></tr><tr><td>Temperature Controlled-Inverted confocal w/458 and 515 nm laser sources&amp;nbsp;</td><td>Zeiss</td><td>&amp;nbsp;LSM 710-w/spectral META detector</td><td></td></tr><tr><td>Outgrowth Media</td><td>Newengland Biolabs</td><td>B9020s</td><td></td></tr><tr><td>NIH 3T3 Fibroblasts</td><td>ATCC</td><td>CRL-1658</td><td></td></tr></tbody></table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

<p class='jove_content'>The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.</p>

<p class="jove_content" fo:keep-together.within-page="1" style=";text-align:right;direction:rtl"> בעקבות בפרוטוקול לעיל, פלסמיד דנ&quot;א נרכשה מהמאגר דנ&quot;א להפוך לתאי <em>חיידק.</em> <em>E. coli</em> להביע הדנ&quot;א חיישן נבחרו מתוך LB / צלחות אמפיצילין ו מוגבר במרק LB נוזלי. בעקבות ההגברה של הווקטורים, פלסמידים DNA היו מטוהרים לתוך חיץ טריס-EDTA באמצעות תקן, ערכת ב?...

Watch this Scientific Journal Video about A Protocol for Using FÃ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

<p class='jove_content'>A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. </p>

פרוטוקול באמצעות העברת אנרגיה פורסטר תהודה (סריג) חיישנים ביולוגיים -force למדוד כוחות מכניים ברחבי המכלול הגרעיני LINC

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

a-protocol-for-using-frster-resonance-energy-transfer-fret-force

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.5K Views.  Virginia Commonwealth University. A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. 

Video: A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Metal Flame Emission

<h4><strong>Metal Flame Emission</strong></h4>
<p>Flame testing is an analytical technique where a sample is placed in a flame, and the characteristic flame color is used to identify the substance. Each element has a characteristic light emission when placed into a flame, meaning each element produces a unique color. This phenomenon is used in fireworks displays, where the color of the fireworks corresponds to a specific characteristic of a metal. The emission of colored light from a burning sample is the direct result of the metallic component absorbing energy due to the excitation from the flame and emitting light as a result.</p>
<h4><strong>Electron Excitement</strong></h4>
<p>When an atomic or molecular species absorbs energy, the energy is used in four different ways. First, the energy is used in translation, causing the molecules to move faster. Second, the energy is used in vibration, which causes the distance between the species to change rapidly. Third, the energy causes rotation, which induces the rotation of atoms around the bonds in the molecule. Finally, the absorbed energy results in electron excitement, which causes electrons to move to a higher energy level from the most stable or ground state.</p>
<p>According to the Bohr model of hydrogen, electrons in an atom exist in discrete states, which correspond to individual shells or orbitals around the nucleus. The lowest energy state is called the ground state and is represented by the notation of n = 1. Excited energy states have higher energies and are represented by the notation of n = 2, 3, 4, etc.</p>
<p>For electrons to move to a higher energy state, they must absorb an amount of energy that is equal to the difference between its ground state and the higher energy state. For example, if an electron absorbs an amount of energy that is equal to the difference between the ground state and the n=3 energy level, the electron will move to the n=3 energy level. An electron can spontaneously relax back down to the ground state or any other lower energy level. When this occurs, a photon is emitted, releasing the absorbed energy.</p>
<p>The released energy is emitted in the form of light. The emitted light has a characteristic energy, and therefore, wavelength, that correlates to the energy levels of the atom. Visible light, which is the light humans can see with their eyes, ranges from about 400 nm to 700 nm on the electromagnetic spectrum.</p>
<p>Atomic absorptions and emissions are discrete wavelengths, called lines. These lines are unique characteristics of an element, like a barcode, and can be used to identify the element.</p>
<h4><strong>Metal Flame Emission Test</strong></h4>
<p>In the metal flame emission test, a metal sample is placed in a flame. The flame provides the energy to excite electrons to a higher energy level. As the electrons relax back down to the ground state, light is emitted with a specific energy relative to the energy levels of the atoms in the sample. Since different atoms have different energy levels, the energy absorbed and emitted from a sample, and thus the wavelength, is specific to the sample.</p>
<p>Metals have characteristic atomic emission wavelengths in the visible range that are easily determined by visual inspection. For example, lithium emits a red color, sodium emits a yellow color, potassium emits a pink-purple color, and barium emits a yellow-green color.</p>
<p>While atomic emissions are discrete wavelengths or lines, most samples of metals contain not only the metal, but also various metal ions, oxides, and salts. Since each atom absorbs and emits a characteristic wavelength of light, the absorbed and emitted light from the flame test contains a range of wavelengths. Thus, atomic absorption and emission spectra can be measured for the sample using a spectrophotometer.</p>
<p>The wavelengths and shapes of the spectra are unique for each substance. For example, the relative intensity of features in the spectrum depends on the concentration of the species. The absolute intensities are dependent on the distance from the sample to the spectrophotometer.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). <em>Chemistry and Chemical Reactivity</em>. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). <em>Chemistry: The Molecular Nature of Matter and Change.</em> Boston, MA: McGraw Hill.</li>
	<li style="font-size:17px">Harris, D.C. (2015). <em>Quantitative Chemical Analysis. </em>New York, NY: W.H. Freeman and Company.</li>
</ol>

פליטת להבת מתכת

Metal Flame Emission
Flame testing is an analytical technique where a sample is placed in a flame, and the characteristic flame color is used to identify ...

Education

JoVE Lab Manual

Chemistry

DNA Isolation and Restriction Enzyme Analysis

<p>The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.</p>

<h4>Isolating DNA from Cells</h4>

<p>DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na<sup>+</sup> ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.</p>

<h4>Quantifying DNA</h4>

<p>Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.</p>

<h4>Restriction Enzymes</h4>

<p>For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3&#8217;-5&#8217; and 5&#8217;-3&#8217; directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3&#8217;...GAATTC&#8230;-5&#8217;, which reads the same in the 5&#8217;-3&#8217; direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.</p>

<p>Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes<sup>1</sup>.</p>

<p>DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases<sup>2</sup>. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions<sup>3</sup>. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual&#8217;s genetic data, paving the way to effectively treat patients without detrimental side effects<sup>4</sup>.</p>

<h4>References</h4>

<ol class="references">
	<li><strong>Chambers, GK, et al.</strong> DNA fingerprinting in zoology: past, present, future. <em>Investig Genet. </em>2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.</li>
	<li><strong>Roewer, L.</strong> DNA fingerprinting in forensics: past, present, future. <em>Investig Genet. . </em>2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.</li>
	<li><strong>Kirkpatrick, BE and Rashkin, MD.</strong> Ancestry Testing and the Practice of Genetic Counseling. <em>J Gen Counseling. </em>2016, Vol. 26, 1 (6-20).</li>
	<li><strong>Hamburg, MA and Collins, FS.</strong> The Path to Personalized Medicine. <em>N Engl J Med . </em>2010; , Vol. 363, 301-304.</li>
</ol>


ניתוח אנזימי בידוד והגבלה של DNA

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Biology

Genetics

Energy Dynamics

<h4>The Food Chain</h4>
<p>Energy is one of the most important abiotic factors in an ecosystem and organisms in an ecosystem are connected by the flow of energy and matter among one another. Since energy can be neither created nor destroyed, it can only change form or be transferred to the next organism in a food chain. For example, every time a cow grazes on grass or an osprey hunts and consumes fish, energy is transferred from the consumed organism to the consumer. Each of these interactions in a food chain is called a trophic level. Energy gained from these food sources is used to build the tissues of these consumers which, in turn, become sources for the next organisms in the food chain. Understanding the dynamics of energy flow in an ecosystem provides a clearer picture of the delicate balance of our natural world.</p>
<p>At the base of the ecosystem, primary producers unlock the energy for the rest of the organisms in the environment. Primary producers are autotrophic or self-feeding organisms because they can synthesize organic molecules from inorganic material. Examples of producers include chemosynthetic bacteria and photosynthetic plants. These organisms simply trap the energy from sources such as gases from hydrothermal vents or sunlight into organic molecules to sustain themselves. They then become a resource for consumers, which are heterotrophic organisms that cannot create their own organic materials and obtain them from other organisms. The organisms that get their energy from autotrophs are called primary consumers. Next on the food chain are secondary consumers that can feed on primary consumers. Similarly, consumers that can feed on secondary consumers are called tertiary consumers.</p>
<p>Energy flow in a food chain starts with the primary producers, thus the size of the community depends on the amount of energy captured into organic material by the primary producers. Organic material stored in an organism is called the biomass and excludes the water contained by the organism. Therefore, to calculate biomass, the weight of water of an organism is subtracted from its total weight. Biomass within a food chain is partially conserved and generally only a fraction of biomass is transferred to the next trophic level. Therefore, the amounts of biomass passed down in a food chain resemble a pyramid, greatest at the bottom, gradually shrinking towards the top. In such a trophic or energy pyramid, the amount of biomass decreases gradually due to the loss of energy as metabolic heat. Hence, the large fraction of energy consumed, but not turned into biomass, indicates how organisms must work to maintain themselves. Respiration is an exothermic reaction that works to power each individual cell by breaking down nutrients to capture the energy into adenosine triphosphate (ATP), which powers the synthesis and transfer of structures and proteins within the cell. Waste products and heat are produced at the same time, resulting in a smaller amount of biomass in the upper-level organism.</p>
<h4>Productivity of an Ecosystem</h4>
<p>Changes in biomass in a system is related to the productivity of a particular ecosystem, where productivity is the rate at which organisms gain biomass from received energy. Productivity of primary producers is called primary productivity and that of others is called secondary productivity.  This can be further categorized into two forms: gross and net.  For instance, gross primary productivity is the rate at which photosynthesis or chemosynthesis occurs, while net primary productivity is the rate at which energy is stored as biomass in these organisms. One can think of net primary productivity as the gross primary productivity subtracted by the energy lost via metabolic processes and daily activities of the organism. Allocation of these biomass resources varies across organisms and indicates the limits of energy supply.</p>
<h4>Human Impacts on Energy Flow</h4>
<p>The trophic pyramid model of energy flow underscores the importance of the primary producers to the health of the ecosystem: If they are removed from a system, the consumers that rely on them must be forced to turn to another food source. If the consumers are unable to find another source of nutrition, secondary extinctions can occur<sup>1</sup>. This is especially important in the near future as human-induced changes will cause unprecedented variations in numerous ecosystems around the world<sup>1</sup>. Therefore, understanding energy dynamics in food chains that are under threat can help mitigate negative effects of environmental changes and prevent secondary extinctions.</p>
<p>Human-induced changes can also impact health of organisms at the top of the food chain, including humans. One well-known example is bioaccumulation and biomagnification of mercury in aquatic food chains. Bioaccumulation of mercury begins at the first level, when mercury is absorbed by organisms at the bottom of the food chain. Biomagnification occurs when mercury is passed down to consumers with its concentration increasing at each trophic level. Finally, various large fish species that are higher on the food chain may contain high levels of mercury<sup>2</sup>. Therefore, health professionals advise against consumption of large quantities of certain fish species.</p>
<p>While loss of a primary producer can be detrimental, elimination of certain consumers called &#8220;keystone predators&#8221; can also have a negative impact. In an experiment where ochre sea stars were removed from a rock, the mussels that these sea stars preyed on overpopulated and exacerbated environmental resources and free space<sup>3</sup>. As keystone predators, the ochre sea stars kept the mussel population in check and helped keep diversity thriving on the rock. When energy accumulated as biomass in the form of mussels, the environment was tipped out of balance. Thus, understanding how energy is transferred in an ecosystem can aid scientists in learning which populations keep others from overgrowing.</p>
<h4>References</h4>
<ol class="references">
	<li><strong>Eklof, A and Ebenman, B.</strong> Species loss and secondary extinctions in simple and complex model communities. <em>J Animal Ecology. </em>2006, Vol. 75, 1 (239-46).</li>
	<li><strong>Gray, JS.</strong> Biomagnification in marine systems: the perspective of an ecologist. <em>Marine Pollution Bulletin. </em>2002, Vol. 45, 46-52.</li>
	<li><strong>Paine, RT.</strong> Food Web Complexity and Species Diversity. <em>The American Naturalist. </em>1966, Vol. 100, 910 (65-75).</li>
</ol>

Studying Energy Dynamics and Productivity - Concept

דינמיקת אנרגיה

The Food Chain
Energy is one of the most important abiotic factors in an ecosystem and organisms in an ecosystem are connected by the flow of energy and ...

Ecology

Cell Structure

<h4>Background</h4>
<p>Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19<sup>th</sup> century, the Cell Theory was proposed to define a cell, which states:</p>
<ul>
	<li>Every living organism is made up of one or more cells.</li>
	<li>The cells are the functional units of all organisms.</li>
	<li>All cells arise from preexisting cells.</li>
</ul>
<p>All cells share common features such as having a plasma membrane, a cytoplasm, DNA, and ribosomes. A plasma membrane is a phospholipid bilayer that surrounds the cell. This thin and fluid layer around the cells serves to isolate the cell&#8217;s contents from its environment and regulates the material exchange with its environment, while also facilitating interactions with other cells. Inside the plasma membrane, the cell is filled with a gel-like fluid called cytoplasm that contains organic molecules, salts, and other materials that are vital for the cell&#8217;s functions. Therefore, biochemical reactions that support life, which are known as metabolic processes, take place inside the cytoplasm. The types of metabolic processes that a cell can execute depend on its genetic information. All cells use DNA as the genetic material, which is the hereditary blueprint to construct cellular structures and products. Finally, all cells use ribosomes to synthesize their protein products.</p>
<p>There are two types of cells based on their genetic material location: prokaryotic, which means &#8220;before nucleus&#8221;, and eukaryotic, which means &#8220;true nucleus&#8221;. Therefore, while both types of organisms have DNA, prokaryotes like bacteria have nucleoids, or &#8220;nucleus-like&#8221; components instead of a nucleus, whereas eukaryotes possess true, membrane-bound nuclei to contain their DNA. Moreover, prokaryotes are relatively small, around 0.1&#8211;5.0 micrometers (&#181;m), in comparison to eukaryotes that can typically range from 10-100 &#181;m in size. The small size of prokaryotes allows quick and effortless distribution of materials within the cell and execution of metabolic processes, as well as swift removal of waste or other products from the cell. Hence, eukaryotic cells possess specialized structures known as organelles, such as mitochondria or Golgi apparatus, to enable the execution of vital functions.</p>
<h4>The Eukaryotic Cell</h4>
<p>The eukaryotic cell is a shared derived trait of all eukaryotes, meaning that it had a single origin that has since been inherited by all eukaryotes. The earliest eukaryotic cells are seen in fossils about 2.4 billion years ago, and are recognizable because they are larger than prokaryotic cells<sup>1</sup>. The origin of this cell type resulted from an endosymbiotic event in which one amoeba-like cell engulfed a micrococcal bacteria and formed a stable coexistence<sup>2</sup>. The engulfed bacteria evolved into the first energy-producing organelles, mitochondria, which are the aerobic metabolism organelles in the cell. Mitochondria have their own separate genome and are similar in size to prokaryotes. They contain two layers of membranes that enclose two distinct compartments. Some of the reactions that break down high-energy biomolecules occur in the inner compartment, whereas the outer compartment houses the reactions that capture the energy released from these compounds into adenosine triphosphate (ATP) molecules to be used as the energy currency of the cell.</p>
<p>Nuclei and mitochondria are not the only shared structures of eukaryotic cells. Other ubiquitous eukaryotic organelles are the smooth and rough endoplasmic reticulum (ER), Golgi apparatus, lysosomes and vacuoles. Endoplasmic reticulum simply means &#8220;network inside the plasma&#8221; and as its name indicates it is a large network of membranes inside the cell, especially around the nucleus. Parts of the rough ER extend from the nuclear membrane and are distinguished from the smooth ER by their rough appearance due to numerous ribosomes on their surface. Rough ER is the site for protein synthesis, such as the proteins that are embedded in the plasma membrane or the proteins that are secreted from the cell. In contrast, smooth ER produces lipid-based products but also contains enzymes to detoxify harmful chemicals. Hence liver cells contain abundant smooth ER. Also, muscle cells contain significant amounts of smooth ER because of the calcium-storing function of this organelle, which is essential for muscle contraction. The Golgi apparatus sorts, modifies, and packages cellular products inside vesicles, which fuse with the plasma membrane to release the products. Some of the proteins that are produced in the rough ER are intracellular digestive enzymes. These enzymes are packed in the Golgi apparatus into special vesicles called lysosomes. The major function of the lysosomes is to digest the food particles engulfed by the cell as well as old cell parts. Vacuoles are sacs of cell membrane that serve as storage units within cells. They may serve to store water to regulate the cell&#8217;s water content as well as store metabolic products or even poisonous molecules, depending on the cell type and the organism.</p>
<h4>Kingdom-specific Organelles</h4>
<p>Eukaryotic cells also developed distinct organelles, specific for each kingdom. For instance, kingdom Plantae and Animalia are both eukaryotic, however the organelles of plant and animal cells differ in key ways that allow them to carry out their lives as producers and consumers, respectively. Terrestrial plants need to grow tall and have rigid stems to hold leaves, which they use for photosynthesis. They also need to be able to retain water taken up by roots. Their cells reflect these specific needs. Unlike animal cells, plant cells have chloroplasts, which are used for photosynthesis and often contain the green pigment chlorophyll. Additionally, they are surrounded by cell walls, which are rigid outer layers made of cellulose to support growth and water retention. Because they need to store large amounts of water to maintain the water pressure in the cell, they have larger vacuoles than animal cells. In addition, plant cells also have another type of specialized storage organelles called plastids, which contain pigments as well as photosynthetic products such as starch. These differences are noticeable and distinguish plant cells from animal cells: plant cells generally have a regular, rectangular shape due to their rigid cell walls, while animal cells are rounded and more irregular.</p>
<h4>Microscopy</h4>
<p>Some cells, such as frog oocytes, are large enough to be seen with the naked eye, yet most cells cannot be seen without any visual aid. Therefore, scientists use microscopy techniques to study cell structures and distinguish cell types from one another. While microscopes are able to magnify objects that are difficult or impossible to be see with the human eye, most tissues naturally lack pigmentation. Therefore, solutions have been created that can selectively stain cells based on their molecular composition. This allows researchers to distinguish between organelles in a cell, tissue types in a plant stem, and fat layers in animals, just to name a few examples. Methylene blue dye stains nucleic acids of dead cells, binding to negatively charged DNA. Safranin solution is another biological dye that stains cell nuclei red. Cells only need to be in the staining solutions for a short period of time, and can be mounted immediately following the staining step. The commonly used mounting techniques are wet mount and oil immersion. A wet mount is created by collecting a sample and putting it on a glass slide with liquid between the slide and the coverslip. Cell samples are suspended in liquids like water or glycerol. Glycerol is better to use with live cultures, because it prevents the proliferation of bacteria<sup>3</sup>. Immersion oil can be added on top of the coverslip to improve viewing of the sample at high magnification. This is accomplished because the oil has the same refractive index as glass, which means that it allows light to pass through it as well as glass would. The glass-air interface scatters light more than oil or glass, so the clarity of the image is affected when samples are mounted &#8220;dry&#8221;, or without oil. Once the cells are stained and mounted, they are ready to be studied under the microscope.</p>
<p>There are various microscopy techniques from electron scanning technology that has allowed researchers to view objects on an atomic level to fluorescent live cell imaging that allows real-time monitoring of the movement of molecules within individual cells<sup>4</sup>. Bright-field microscopy is the simplest microscopy technique, requiring only a halogen source light, a condenser lens to focus the light, an ocular lens to view the image, and an objective lens to magnify the image. With any microscopy technique, it is important to understand the parts of a microscope before using one. In general, compound microscopes used for bright-field imaging have an eyepiece at the top of the scope, which is attached to the head and objectives. The eyepiece has a magnification of 10X, and objective lenses are set to a particular magnification in a range of 4X-100X. There are between three and five objectives on a standard microscope. The objectives point down to the stage, which is where a specimen is placed for viewing. The stage often has mechanical parts and stage clips to hold a slide and move it around while viewing. An aperture is a hole in the stage for light to pass through. This light is controlled by an adjustable condenser lens above an illuminator, or light source. To control the zoom of the stage for the object being viewed, microscopes feature coarse and fine focus adjustment knobs. The coarse focus knob moves on a larger scale than the fine focus, but they are on the same axis. The fine focus is useful when the object on the stage has been brought close to the objectives. It is important not to let the objective lens touch the object on the stage, as it can scratch the lens. Objects should always be first viewed on the lowest magnification objective and clearly focused before switching to higher magnification objectives.</p>
<p>Microscopy is an important tool for many aspects of the medical field including research, diagnosis, and treatment. This has the application of using nanotechnology in medicine, as a new treatment method in place of a more invasive surgery<sup>5</sup>. Surgeons also make use of microscopes, some of which have been modified to be mounted on a surgeon&#8217;s head and are operated with foot pedals. These are of much lower magnification than even the light microscopes used today, but they facilitate the safe execution of delicate procedures, such as optical and neurosurgery.</p>
<h4>References</h4>
<ol class="references">
	<li><strong>Bengtson S, Rasmussen B, Ivarsson M, Muhling J, Broman C, Marone F, Stampanoni M, Bekker A.</strong> Fungus-like mycelial fossils in 2.4-billion-year-old vesicular basalt. <em>Nature Ecology &#38; Evolution. </em>2017, Vol. 1, Article number: 0141.</li>
	<li><strong>Vellai T, Vida G.</strong> The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells. <em>Proc. R. Soc. Lond. B. </em>1999, Vol. 266, 1571-1577.</li>
	<li><strong>Gouet V, Roger G, Fonty C, Andre P.</strong> Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi. <em>Current Microbiology. </em>24, 1992, Vol. 4, 197-201.</li>
	<li><strong>Cognet L, Leduc C, Lounis B.</strong> Advances in live-cell single-particle tracking and dynamic super-resolution imaging. <em>Curr Opin Chem Biol. </em>2014, Jun; 20:78-85.</li>
	<li><strong>Asiyanbola B, Soboyejo W.</strong> For the surgeon: an introduction to nanotechnology. <em>J Surg Educ. </em>2008, Vol. 65, 2 (155-61).</li>
</ol>

Cell Structure: Visualizing Onion and Human Cells - Concept

מבנה התא

Background
Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, ...

Fundamentals

Macromolecules

<h4>Biomolecules</h4>
<p>Organisms contain a wide variety of organic molecules with numerous functions which depend on the chemical structures and properties of these molecules. All organic molecules contain a carbon backbone and hydrogen atoms. The carbon atom is central in the formation of a vast variety of organic molecules ranging in size, shape and complexity; inorganic molecules on the other hand, generally have simpler structures. The outermost shell of a free carbon atom can accommodate eight electrons but is occupied by only four electrons, therefore it can form four covalent bonds and bond with up to four atoms. Alternatively, it can also bond with fewer atoms by forming double or triple bonds. This versatility of carbon atoms allows organic molecules to display intricate structures, such as chains, branches, and rings, among others.</p>
<p>Organic molecules that naturally occur in organisms are called biomolecules. Besides carbon and hydrogen, biomolecules also contain other elements such as oxygen, nitrogen, phosphorus, and sulfur. In general, smaller units of biomolecules come together, as repetitive sequences, to form larger biomolecules. These small modular units of biomolecules are called monomers. Two monomers typically join each other to form a dimer through a process known as <em>dehydration synthesis</em>, which is simply the removal of a hydrogen atom from one monomer and a hydroxyl (OH<sup>-</sup>) ion from the other monomer to create a water molecule to be expelled out while linking the two monomers with a covalent bond. The reverse of this process is called <em>hydrolysis</em>, in which the molecule splits back into its original monomers with a water molecule providing a hydrogen atom to one monomer and a hydroxyl ion to the other. Many monomers can be attached together by dehydration to form polymers. Sometimes different polymers can come together to form even larger and more complex molecules, which are known as biological macromolecules.</p>
<p>Biomolecules are classified based on the elements that compose them, and their structure and function inside living organisms. Almost all biomolecules can be classified into one of the four general categories: carbohydrates, lipids, proteins, and nucleic acids.</p>
<h4>Carbohydrates</h4>
<p>Carbohydrate simply means &#8220;carbon water&#8221; because these molecules are composed of carbon, hydrogen, and oxygen atoms roughly in the ratio of 1:2:1. Carbohydrate monomers are known as monosaccharides, which are also referred to as <em>simple sugars</em>. Glucose (C<sub>6</sub>H<sub>12</sub>O<sub>6</sub>) is the most common monosaccharide in living organisms and is a subunit of many polysaccharides. Numerous organisms also synthesize other six-carbon monosaccharides with the same chemical formula as glucose but slightly different structures, such as fructose and galactose. When two monosaccharides are linked together, they form disaccharides. For example, sucrose is composed of glucose and fructose, whereas lactose contains glucose and galactose. These monosaccharides and disaccharides are used for short-term energy storage in living organisms.  Maltose is another disaccharide that is made up of two glucose molecules and is usually formed when polysaccharide chains such as starch and glycogen are broken down during digestion. Starch is a polysaccharide that serves as an energy storage molecule in plants and is made up of two types of glucose polymers: amylose and amylopectin. Amylose constitutes 10-20% of starch and is a helical polymer of glucose. Amylopectin makes up the bulk of the starch and is a branched polymer of glucose. Glycogen is virtually the same as starch, however it is synthesized, stored and used in animal liver and muscle tissues.</p>
<p>Besides serving as energy stores, carbohydrates also have other functions in organisms. The five-carbon monosaccharides, ribose and deoxyribose, are integrated into the nucleic acid structure and are present in every living cell. Moreover, the polysaccharide cellulose, which is a long polymer made up of glucose, serves as a rigid structural material in plants. Humans do not have digestive enzymes to break down cellulose in food, which is also called dietary fiber. However, dietary fiber consumption helps to maintain a healthy gut flora, which in turn contributes to the health of digestive and immune systems<sup>1</sup>. Similar to plants, some animals and fungi use another polysaccharide, chitin, as a structural molecule. Arthropods use chitin to build and maintain their exoskeletons, whereas fungi incorporate it into their cell walls to maintain rigidity.</p>
<h4>Lipids</h4>
<p>The second class of biological macromolecules are lipids, which include fats, oils, and waxes. Lipids are hydrophobic molecules that are almost entirely made up of carbon and hydrogen atoms. Often, lipids are grouped in three major categories; triglycerides, phospholipids, and steroids.</p>
<p>The most common type of lipid is triglycerides, which include fats from animals and oils from plants. Triglycerides generally serve as long-term energy storage molecules, except indigestible waxes, which are instead used as a waterproofing substance in both plants and animals. Triglycerides contain three fatty acid chains, which can be either saturated or unsaturated, connected to a glycerol molecule. Saturated fatty acid chains are linear molecules with a maximum number of hydrogen atoms, where every carbon in the chain is connected via a single bond. On the other hand, unsaturated fatty acid chains have kinks due to the presence of at least one double bond. Additionally, unsaturated fats can be &#8220;trans&#8221; fats if the hydrogens around the double bond oppose each other. While trans fats occur naturally, they are generated during industrial production of saturated vegetable oils with hydrogen. Similar to saturated fatty acids, trans fats stack very well due to their relative linearity. However, trans fats cause problems for human heart health, such as the damaging the lining of arteries and causing inflammation when digested<sup>2</sup>.</p>
<p>Phospholipids are similar to triglycerides, however, one of the fatty acid chains is replaced with a phosphate-containing polar group. Therefore, phospholipids have a hydrophilic head and two hydrophobic fatty acid tails. These properties of phospholipids are crucial to the cell membrane structure and function.</p>
<p>Steroids are lipids that are composed of fused carbon rings with varying functional groups. Cholesterol is a steroid that is also a cell membrane component. Moreover, cholesterol is used to synthesize other steroids, including sex hormones such as estrogen and testosterone. Although cholesterol is essential for cell membrane structure and hormone synthesis, high levels of plasma cholesterol are implicated in plaque accumulation inside blood vessels and causing coronary disease<sup>3</sup>.</p>
<h4>Proteins</h4>
<p>The third class of biological macromolecules are proteins, which are made up of chains of amino acids. There are 20 different amino acids, all with a similar base structure but each has a unique side chain called an &#8216;R-group&#8217;. A single amino acid has an N-terminal end which is an amino group (NH<sub>3</sub><sup>+</sup>) and a C-terminal end which is a carboxyl group (-COOH). These groups link together, N-terminal to C-terminal, in a chain connected by peptide bonds. Proteins are important for maintaining body functions as enzymes, hormones, structural components and transport molecules, and play vital roles in muscle contractibility, immunity and blood clotting. However, issues can arise in protein structure and function, and these issues are often genetic. For instance, normal red blood cells are round, but in people affected by sickle cell anemia, cells have a curved shape with an exposed hydrophobic region, caused by a mutation in a protein called hemoglobin S. This shape reduces the capacity to carry oxygen and causes the cells to get stuck in blood vessels. This results in many detrimental symptoms to the person carrying the mutation, and people who inherit two copies of the sickle cell gene often suffer ill effects or even possibly die because of the reduced capacity of sickled cells to transport oxygen. In a twist, those carrying only one copy of the gene are resistant to infection from malaria, so the disease has been able to be passed on and persists in countries with elevated levels of malaria infections<sup>4</sup>.</p>
<h4>Nucleic Acids</h4>
<p>The fourth class of biological macromolecules are the nucleic acids, which are composed of monomers known as nucleotides. These monomers are composed of three parts: a phosphate group, a ribose sugar and a nitrogen base. Nucleotides differ from each other by their nitrogen bases and the type of ribose they contain.  Single nucleotides generally act as energy carriers inside cells, as well as function as messenger molecules. However, the nucleotide polymers or nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) are hereditary molecules that contain the genetic information to construct cellular products.</p>
<h4>Macromolecule Detection</h4>
<p>Biological macromolecules in food or other substances can be detected by using their specific chemical properties. For example, monosaccharides have free aldehyde (-CHO) or ketone (-C=O) groups that can reduce other compounds, meaning they are substances that cause other molecules to lose electrons, thus monosaccharides are also known as <em>reducing sugars</em>. This property is used to detect the presence of monosaccharides with an indicator called Benedict&#8217;s reagent.  This indicator contains copper ions (Cu<sup>2+</sup>) which are reduced by monosaccharides as observed with a change of the solution color from blue to a reddish orange, though the intensity of the color varies based on the original concentration of the reducing sugar. As such, observation of a green color means that there is only a small amount of reducing sugars present in the solution. Amylose in starch exists in a coiled structure because of the bond angles in the polymer chain. The iodine indicator, iodine-potassium iodide (IKI), reacts with these coiled molecules and will turn the solution a dark bluish-black to indicate the presence of amylose. However, if starch amylose isn&#8217;t present, the reaction will not take place, and the solution will remain a yellowish-brown color.  A Sudan IV test is performed to test for the presence of lipids. This dye is lipophilic and solubilizes when lipids are present, thus a red color is retained in the presence of lipids. Biuret&#8217;s reagent, an indicator for the presence of proteins, contains copper ions (Cu<sup>2+</sup>) which react with the peptide bonds and turn the solution from blue to dark violet color. This reagent needs to react with a sufficient number of these peptide bonds to produce the expected violet color, so a pinkish color will result if the amino acid chains are not long enough.</p>
<h4>References</h4>
<ol class="references">
	<li><strong>Rasnik K. Singh, Hsin-Wen Chang, Di Yan, Kristina M. Lee, Derya Ucmak, Kirsten Wong, Michael Abrouk, Benjamin Farahnik, Mio Nakamura, Tian Hao Zhu, Tina Bhutani, Wilson Liao.</strong> Influence of diet on the gut microbiome and implications for human health. <em>J Transl Med. </em>2017, Vol. 15, 73 doi: 10.1186/s12967-017-1175-y.</li>
	<li><strong>Siri-Tarino PW, Sun Q, Hu FB, Krauss RM.</strong> Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. <em>Am J Clin Nutr. </em>2010, Vol. 91, 3 (535-46).</li>
	<li><strong>Joseph L. Goldstein, Michael S. Brown.</strong> A Century of Cholesterol and Coronaries: From Plaques to Genes to Statins. <em>Cell. </em>2015, Vol. 161, 1 (161-72).</li>
	<li><strong>Michael Aidoo, Dianne J Terlouw, Margarette S Kolczak, Peter D McElroy, Feiko O ter Kuile, Simon Kariuki, Bernard L Nahlen, Altaf A Lal, Venkatachalam Udhayakumar.</strong> Protective effects of the sickle cell gene against malaria morbidity and mortality. <em>Lancet. </em>2002, Vol. 359, 9314 (1311-1312).</li>
</ol>

Macromolecules: Identification Using Indicator Solutions - Concept

מקרומולקולות

Biomolecules
Organisms contain a wide variety of organic molecules with numerous functions which depend on the chemical structures and properties of these ...

UV-Vis Spectroscopy of Dyes

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Dye Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">First, put on a lab coat, safety glasses, and nitrile gloves. Some solutions use hexane or dimethylformamide, so prepare those solutions in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare a 1 mM stock solution of fluorescein in water. Measure 0.0166 g of fluorescein and place it in a 50-mL volumetric flask.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Add about 30 mL of deionized water to the flask, stopper it, and swirl it until as much solid has dissolved as possible. Then, fill the flask to the line with deionized water.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Stopper the flask, wrap the top in plastic paraffin film, and invert it several times while firmly holding the stopper in place to thoroughly mix the solution. Label the flask with the solution&#39;s name and concentration and wrap it in aluminum foil to protect the dye from light. Label the outside of the foil as well.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare a 3 &#181;M fluorescein test solution from the stock solution. Use a micropipette to transfer 150 &#181;L of the stock solution to a clean 50-mL volumetric flask. Add about 30 mL of deionized water and swirl the flask to mix them.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill the flask to the line with deionized water, stopper and seal it, and invert the flask several times until the solution is well mixed. Label the flask with the solution&#39;s name and concentration, wrap it in foil, and label the outside of the foil as well.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Prepare 1 mM stock solutions and 3 &#181;M test solutions of indigo in dimethylformamide and &#946;-carotene in hexane in the same way. The masses of solid &#946;-carotene and indigo that you will need to make the solutions are listed in the tables.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td colspan="4" style="border: 1px solid; padding: 0px; text-align: center;"><strong>All stock solutions in 50 mL solvent</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Dye</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Stock concentration </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Mass</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Solvent </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1 x 10<sup>-3 </sup>(1 mM)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.027 g</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Hexane</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Indigo</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1 x 10<sup>-3 </sup>(1 mM)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">0.013 g</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">DMF</td>
				</tr>
			</tbody>
		</table>
		<ul style="width:100%;">
			<li style="width:100%;"></li>
		</ul>
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td colspan="4" style="border: 1px solid; padding: 0px; text-align: center;"><strong>All test solutions diluted to 50 mL </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Dye</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Test concentration </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Stock volume </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Solvent </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3 x 10<sup>-6 </sup>(3 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">150 &#181;L</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Hexane</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">Indigo</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3 x 10<sup>-6 </sup>(3 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">150 &#181;L</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">DMF</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use the stock solution of &#946;-carotene to make five more solutions of the concentrations listed in this table. Wrap the flasks in foil afterward.
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td colspan="3" style="border: 1px solid; padding: 0px; text-align: center;"><strong>All solutions diluted to 50 mL with hexane</strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Dye</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Concentration</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Stock volume </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">1.9 x 10<sup>-6 </sup>(1.9 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">95 &#181;L</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">3.7 x 10<sup>-6 </sup>(3.7 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">185 &#181;L</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">7.5 x 10<sup>-6 </sup>(7.5 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">375 &#181;L</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">11 x 10<sup>-6 </sup>(11 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">550 &#181;L</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;">&#946;-carotene</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">15 x 10<sup>-6 </sup>(15 &#181;M)</td>
					<td style="border: 1px solid; padding: 0px; text-align: center;">750 &#181;L</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">When you&#39;re finished, you&#39;ll have three micromolar solutions of fluorescein, &#946;-carotene, and indigo dye for the first part of the lab and five &#946;-carotene solutions with different concentrations for the second part of the lab. Store the solutions in a refrigerator for now if you have one. If not, store them at room temperature for up to 24 hours.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Put organic and aqueous waste containers in a fume hood with a glass waste container nearby.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Fill a 250-mL wash bottle with acetone and place it with the waste containers. Next, place the dye solutions and containers of hexane and DMF in the hood.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Fill another wash bottle with deionized water and place it with the solvents.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Set Pasteur pipettes, several pipette bulbs, and lab wipes nearby. Then, place quartz cuvettes in a central area for the students. Place another box of lab wipes by the spectrometer.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">About 10 to 20 min before the lab, turn on the UV-Vis spectrometer and the instrument computer.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Once the spectrometer is ready, configure it so the default scan is absorbance with a range of 200 &#8211; 800 nm.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Create a folder where students can save their spectra and set the default file path to that folder. When the students arrive, explain how to use the software and save their data.</div>
		</li>
	</ul>
	</li>
</ol>

Ultraviolet-Visible (UV-Vis) Spectroscopy of Dyes - Prep

ספקטרוסקופיית UV-Vis של צבעים

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of Dye SolutionsExpand
	Here, we show the laboratory preparation ...

Lab Manual Sub Articles

Synthesis of Luminol

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. The chemicals that you will use are hazardous, so both solutions must be prepared and handled in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 10 mL of a 10% by volume solution of hydrazine in water. Measure 8.4 mL of deionized water and add it to a 30-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 1.6 mL of hydrazine hydrate and pour it into the beaker for a total volume of 10 mL. Stir the hydrazine solution with a glass rod until it is well mixed. <strong>Note:</strong> Hydrazine is flammable, toxic, and explosive.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle. Cap it and label it with the solution&#39;s name and concentration. Store the hydrazine hydrate and the hydrazine solution in a cabinet for flammables.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 20 mL of 2.8 to 3 M aqueous NaOH. <strong>Note:</strong> NaOH is highly corrosive. Avoid skin contact.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Measure 2.2 to 2.4 g of solid NaOH and add it to a 50-mL beaker. Add 20 mL of deionized water and stir the mixture on a stir plate until the NaOH has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Pour the solution into a 20-mL glass bottle and close it with a lid. Label the bottle with the solution&#39;s name and approximate concentration. Store the NaOH pellets and solution in the appropriate cabinets.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Lastly, clean your glassware following your usual procedures.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Before you set up the main lab, reserve a separate room that you can make as dark as possible.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Prepare a container for fluorescein waste and ensure that a glass waste container is available.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out a 10% sodium hypochlorite (bleach) solution and a 1 M sodium carbonate solution in the waste hood.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Fill a wash bottle with deionized water and place it in the same hood. Confirm that there are paper towels and a test tube brush by each sink in the main lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring containers of 3-nitrophthalic acid, anhydrous sodium dithionite, solid potassium hydroxide, and fluorescein to the balance area.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Confirm that there are enough laboratory wipes, spatulas, and weighing boats for the class.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set triethylene glycol, the hydrazine solution, the NaOH solution, glacial acetic acid, and dimethyl sulfoxide in a central hood. Place a 1-mL graduated pipette and a pipette controller designated for hydrazine in the same hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Put Pasteur pipettes, filter papers, scissors, and plastic paraffin film in a central area.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stirring hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Bunsen burner with tubing</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Lab stands</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;3&#160;&#160;&#160;&#160;Clamps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Pair of flask tongs</td>
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					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Medium filter adapter</td>
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					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Test tube stopper</td>
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					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small flask stopper</td>
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					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Fire striker or lighter</td>
				</tr>
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					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape or labels</td>
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		</li>
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	</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Prep

סינתזה של לומינול

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of SolutionsExpand
	Here, we show the laboratory preparation for ...

Beer's Law

Beer-Lambert Law and Equilibrium Constant Determination - Procedure

חוק הבירה

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Lab Setup for Beer&#8217;s LawExpand
	In this lab, you will combine aqueous solutions of FeCl3 ...

Enzyme Activity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Investigating the Effect of pH and Temperature on Peroxidase Activity
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To make the extraction buffer for the peroxidase enzyme, mix equal volumes of 0.1 M sodium phosphate monobasic and 0.1 molar sodium phosphate dibasic solutions to achieve a final volume of 500 mL.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Test the resulting solution using a pH meter. The mixture should be between a pH of 6.8 and 7.2.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">To extract the turnip peroxidase, use a knife to remove the outer layer of a turnip and then cut the turnip into approximately two cm cubes.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place 30 g of turnip cubes into a blender with 300 mL extraction buffer, and blend the turnip cubes at high speed three times for about 15 s each time.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Filter the homogenized mixture through three layers of cheesecloth, then strain the resulting solution through one coffee filter.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Store the filtrate in a 500 mL bottle, and immediately place the bottle in the refrigerator.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, to make the enzyme substrate solutions, first add 15 mL 3% H<sub>2</sub>O<sub>2</sub> to 435 mL distilled water to dilute the H<sub>2</sub>O<sub>2</sub> to a 0.1% concentration.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">To prepare the color-changing reactant, dissolve 0.2 mL concentrated guaiacol in 100 mL of isopropyl rubbing alcohol, and store this guaiacol solution in the refrigerator.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, to make solutions for the different pH conditions, add 100 mL distilled water to six 250 mL beakers, and place the buffer capsule contents with pH of 3, 4, 5, 6, 7, and 8 into separate beakers.
</div></li></ul></li></ol>

Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Prep

פעילות אנזימים

Investigating the Effect of pH and Temperature on Peroxidase Activity
ExpandTo make the extraction buffer for the peroxidase enzyme, mix equal volumes of ...

Cellular Processes

Cellular Respiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Quantifying Respiration using Microrespirometers
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To assemble the microrespirometers needed for the lab, first plug in the hot glue gun to preheat it.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Push the plunger of a tuberculin syringe all the way in and then cut the end off of the syringe.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, insert a capillary tube into the empty needle end of the syringe.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Push the capillary tube in until it touches the tip of the plunger.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Use the hot glue gun to create an airtight seal between the capillary tube and the tuberculin syringe. Make sure that the capillary tube is standing straight up while the glue dries.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Once the glue has dried, pull back on the plunger and make sure the capillary tube is not plugged.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Next, prepare the manometer fluid by adding red food coloring to soapy water. NOTE: The detergent coats the inside of the capillary tube, preventing the manometer fluid from sticking during the experiment.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Insert the capillary tube into the manometer fluid and then pull the plunger on the syringe until the entire capillary tube has been filled.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, eject the manometer fluid back out of the microrespirometer.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">With a small piece of absorbent cotton, plug the barrel of the microrespirometer, inserting it into the syringe up to the 0 mL mark. Pack the cotton using a spare capillary tube or a glass disposable pipette.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Add a single drop of 15% KOH to the cotton using a spare capillary tube or a glass disposable pipette.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Add some nonabsorbent cotton to the KOH saturated cotton to act as a barrier to the seeds. Pack the cotton using a disposable pipette. The cotton should reach halfway between the 0 mL and 1 mL mark.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Reinsert the plunger carefully until it reaches the cotton and then remove any excess KOH by pressing it out.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Set four microrespirometers at each lab station.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Then, place a lab marker and two small dishes containing the seeds and glass beads for each group alongside the microrespirometers.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Finally, prepare two water baths by filling them with roughly 6 inches of water. The water level in the bath should comfortably fit two respirometers with the entirety of their two capillary tubes exposed.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Replace the lids and insert a thermometer in each bath to monitor the temperature. One bath should be set at about 25 &#176;C, and the other at approximately 40 &#176;C.
</div></li></ul></li></ol>

Cellular Respiration: Quantification using Microrespirometers - Prep

נשימה תאית

Quantifying Respiration using Microrespirometers
ExpandTo assemble the microrespirometers needed for the lab, first plug in the hot glue gun to preheat ...