بتمويل من المعاهد الوطنية للصحة HL56621 R01.

العدلة عزل بروتوكول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع كل الكواشف إلى درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> جمع 5.0 مل من وسائل الإعلام في عزلة العدلة أنبوب الطرد المركزي. مل بعناية 5.0 طبقة من الدم على وسائل الإعلام الانفصال. تنفيذ هذه الخطوة ببطء وبعناية ، ومع طرف ماصة على مقربة من سطح وسائل الإعلام إلى تجنب الاختلاط الدم ووسائل الإعلام. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في إطار التعاون الإقليمي ل500 35 دقيقة في 20-25 درجة مئوية. ينبغي للفصل الدم إلى 6 فرق متميزة : البلازما ، وحيدات ، ووسائل الإعلام العزلة ، والعدلات ، وسائل الاعلام مزيدا من العزلة ، وخلايا الدم الحمراء بيليه (الشكل 1). إذا كانت هذه العصابات ليست واضحة ، كانت عملية الفصل ليست نظيفة وستكون هناك حاجة لتكرارها. </li><li style=";text-align:right;direction:rtl"> إزالة بعناية أكبر ثلاث طبقات (البلازما ، وحيدات ، ووسائل الإعلام العزلة) باستخدام ماصة. التخلص من هذه الطبقات. </li><li style=";text-align:right;direction:rtl"> ماصة بعناية طبقة من العدلات وجميع وسائل الإعلام عزلة تحت العدلات. وضع الحل في أنبوب الطرد المركزي نظيفة. </li><li style=";text-align:right;direction:rtl"> تمييع الحل العدلة إلى 10 مل مع HBSS دون كا 2 + / + 2 ملغ. عكس الأنبوب عدة مرات لوقف الخلايا. </li><li style=";text-align:right;direction:rtl"> الحل العدلة الطرد المركزي في 350 RCF لمدة 10 دقائق. وينبغي أن يكون حاضرا بيليه حمراء في أسفل الأنبوب ، وتحتوي على العدلات المتبقية خلايا الدم الحمراء (كرات الدم الحمراء). إزالة طاف مع ماصة بعناية بحيث لا بالانزعاج بيليه. </li><li style=";text-align:right;direction:rtl"> لكرات الدم الحمراء وليز المتبقية ، إضافة 2 مل تحلل الخلايا الحمراء العازلة للأنبوب. لresuspend ، وبيليه دوامة القارورة في وضع 3-4. تجنب زيادة الإعداد دوامة 4 أعلاه ، لأن هذا قد يؤدي إلى تفعيل العدلات. قد يكون من الضروري دوامة لعدة ثوان ، أو إلى &quot;نبض&quot; الدوامة حل لبيليه. </li><li style=";text-align:right;direction:rtl"> أنبوب الطرد المركزي في 250 RCF لمدة 5 دقائق. إزالة طاف مع ماصة. كرر العملية lysing إذا لزم الأمر. </li><li style=";text-align:right;direction:rtl"> إضافة 500 HBSS ميكرولتر دون كا 2 + / + 2 ملغ لكل أنبوب. مرة أخرى ، إلى دوامة resuspend الكرية في وضع 3-4. مخفف الى 10 مل مع HBSS دون كا 2 + / + 2 ملغ. </li><li style=";text-align:right;direction:rtl"> أنابيب الطرد المركزي في 250 RCF لمدة 5 دقائق. نضح في وطاف تجاهل. </li><li style=";text-align:right;direction:rtl"> Resuspend وبيليه في 250 HBSS ميكرولتر / الحل HSA (2 ٪ HSA). ثم الخلايا قد تحسب وتعديلها لتركيز المطلوب. </li></ol>

<ol>
	<li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+leucocytes+from+blood+and+bone+marrow.">Separation of leucocytes from blood and bone marrow.</a> Scand. J. Clin. Invest. 21, 77-83 (1968).</li><li>England, J. M., Rowan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assignment+of+values+to+fresh+blood+used+for+calibrating+automated+blood+cell+counters.">The assignment of values to fresh blood used for calibrating automated blood cell counters.</a> Clin. Lab. Haemat. 10, 203-212 (1988).</li><li>Garcia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+leukocyte+extraction+methods+for+cytomegalovirus+antigenemia+assay.">Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.</a> J. Clin. Microbiol. 34, 182-184 (1996).</li><li>Ferrante, A., Thong, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+conditions+for+simultaneous+purification+of+mononuclear+and+polymorphonuclear+leucocytes+from+human+peripheral+blood+by+the+Ficoll-Hypaque+method.">Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method.</a> J. Immunol. Methods. 36, 109-109 (1980).</li></ol>

يتم استخدام أسلوب التدرج كثافة الفصل لعزل العدلات الإنسان من الدم الكامل باستخدام مزيج من الصوديوم وmetrizoate ديكستران 500. ويستند هذا الأسلوب على طريقة فصل الكريات البيضاء وحيدة النواة التي Boyum (1968) الذي تم تعديله من قبل لفصل العدلة فيرانتي وثونغ (1980). بعد ج?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Lymphocyte Poly(R)</td>
<td>Reagent</td>
<td>Cedarlane Labs</td>
<td>&nbsp;</td>
<td>PolymorphprepTM can be used as an alternative</td>
</tr>
<tr>
<td>Hank's Balanced Salt Solution without Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum Albumin</td>
<td>Reagent</td>
<td>ZLB Bioplasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Red Cell Lysis Buffer</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortexer</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Pipettes and bulb</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PolymorphprepTM</td>
<td>Reagent</td>
<td>Axis-Shield PoC</td>
<td>&nbsp;</td>
<td>Alternative reagent to Lymphocyte-Poly (R)</td>
</tr>
<tr>
<td>Syringe Filter</td>
<td>Other</td>
<td>Millipore</td>
<td>SLGV033RS</td>
<td>Millex-GV, 0.22 &#181;m, PVDF, 33 mm, gamma-sterilizable</td>
</tr>
<tr>
<td>Hank's Balance Salt Solution with Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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neutrophil isolation protocol

المحببة النوى العدلة (PMN) هي الكريات البيض الأكثر وفرة في البشر وبين الخلايا الأولى للوصول في موقع رد الفعل المناعي للالتهابات. نظرا لدورها الرئيسي في الالتهاب ، وتدرس على نطاق واسع وظائف العدلات مثل الحركة والإنتاج خلوى ، البلعمة ، ومكافحة الأورام الخلية. لتوصيف وظائف محددة من العدلات ، وهي طريقة نظيفة وسريعة وموثوقة للفصل بينها وبين غيرها من خلايا الدم غير المرغوب فيه في الدراسات المختبرية ، وخصوصا العدلات قصيرة الأمد ويجب استخدامها خلال 2-4 ساعات من جمعها. هنا ، علينا أن نظهر متدرجة الكثافة الطريقة المعيارية الفصل لعزل العدلات الإنسان من الدم الكامل باستخدام وسائل الإعلام المتاحة تجاريا الانفصال التي هي مزيج من الصوديوم وmetrizoate ديكستران 500. الإجراء يتكون من طبقات الدم كله على مدى الانحدار متوسطة الكثافة ، الطرد المركزي ، والفصل بين طبقة العدلة ، وتحلل من الكريات الحمراء المتبقية. ثم يتم غسل الخلايا ، وعدها ، ومعلق في المخزن الى التركيز المطلوب. عندما يقوم بشكل صحيح ، وقد تبين هذا الأسلوب لعينات من محصول&gt; العدلات 95 ٪ مع بقاء&gt; 95 ٪.

Watch this Scientific Journal Video about Neutrophil Isolation Protocol at JoVE.com

Neutrophil Isolation Protocol

العدلات هي من بين الخلايا الأولى للوصول في موقع رد الفعل المناعي للالتهابات ، وخضعت للدراسة وظائفها وآليات على نطاق واسع في المختبر. نظهر متدرجة الكثافة الطريقة المعيارية الفصل لعزل العدلات الإنسان من الدم الكامل باستخدام وسائل الإعلام فصل متاحة تجاريا.

العدلة عزل بروتوكول

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

101.9K Views.  University of Pennsylvania. العدلات هي من بين الخلايا الأولى للوصول في موقع رد الفعل المناعي للالتهابات ، وخضعت للدراسة وظائفها وآليات على نطاق واسع في المختبر. نظهر متدرجة الكثافة الطريقة المعيارية الفصل لعزل العدلات الإنسان من الدم الكامل باستخدام وسائل الإعلام فصل متاحة تجاريا.

Video: Neutrophil Isolation Protocol

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

بروتوكول للتربية البعوض (A. الغامبية)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

مضان في الموقع التهجين (FISH) البروتوكول في الحيوانات المنوية البشرية

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

بروتوكول لإنتاج Tetramer KLRG1

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

الحمض النووي القائم على تحديد الأنواع السمكية البروتوكول

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

بروتوكول SIVQ-LCM لالصك ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

بروتوكول لعزل خلايا الكبد الإنسان الابتدائية والموافق السكان الرئيسية للخلايا الكبد غير متني

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

بروتوكول المسح الضوئي على مدار الساعة لتحليل الصور: إيماجيج الإضافات

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

بروتوكول بسيط عالي الكفاءة لعزل الجزيرة البنكرياسية عن الفئران

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

الببتيد40 موب للاستخدام في الكشف عن أحداث التهاب العَدلات بوساطة

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

دهن السريع الحبرية العزلة بروتوكول استخدام عدة عزل عضية الراسخة

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...