We thank Dr. Gilles Dupuis for critical reading of the manuscript. 
Funding
This work was supported by the Canadian Institutes for Health Research, grant # MOP-126173. C.M.D is a member of the Fonds de la Recherche en Sant&#233; (FRSQ)-funded Centre de Recherche Clinique &#201;tienne LeBel.

携帯pHの校正のためのソリューションの1。準備<ol><li>キャリブレーションに使用される5溶液を調製するために5つの別々の50mlコニカルチューブに以下の化合物を追加します。 <ul><li>塩化ナトリウム（1MのNaClの1ミリリットル）（1MのNaCl = 10 mlのH 2 O中0.58グラム） </li><li>塩化カリウム（1 MのKCl 6.75ミリリットル）（1 M KClを10 mlのH 2 0で0.75グラム=）を</li><li>グルコース（1 M D-グルコース1mlの（1M）の10ml H 2 O中のD-グルコース= 1.80 g）を</li><li>硫酸マグネシウム4（0.05ミリリットルの1M硫酸マグネシウム）（10ミリリットルのH 2 O中の1MのMgSO 4 = 1.20グラム） </li><li> 20のHEPES（1MのHEPES 1ミリリットル）（1 M HEPES = 10 mlのH 2 O中2.38グラム） </li><li> 塩化カルシウム（1M CaCl 2を 0.05ミリリットル）（10ミリリットルのH 2 O中の1MのCaCl 2 = 1.47グラム） 二重蒸留H 2 O（蒸留H 2 O）のすべてのソリューションを準備します。 </li></ul></li><li>のddH 2 Oを35ミリリットルで、各ソリューションを完成させ、これまで磁気撹拌機で混ぜるlutionが達成される。 </li><li> 5.5、6.0、6.5、7.0および7.5のpH値を得るために、1 N KOHまたは1N HClで各溶液のpHを調整する。その後、50ミリリットルの最終容量を脱イオン水で音量を調整します。 </li><li>対策5ニゲリシンのmgおよびストック溶液ナイジェリシン10 mMのを準備するために無水エタノールを670μLに追加します。 注意：ナイジェリシンは非常に毒性であり、細心の注意（手袋、保護メガネとマスク）で処理する必要があります。ナイジェリシンが完全に溶解するまで反転により溶液を混合する。 </li><li>各校正液に10 mMのナイジェリシン（最終濃度10μM）の0.05ミリリットルを追加します。ニゲリシンは、細胞膜透過性を増加させ、細胞外K +濃度の脱分極の存在下で、細胞内および細胞外pHの平衡化を引き起こすイオノフォアである。 </li><li>較正溶液は、4℃で1ヶ月まで保存することができる</li></ol> 2。細胞の調製<ol><li>生物学的な職場の安全の下でyはフード、10％ウシ胎児血清および抗生物質を補充した増殖培地2mlにある35ミリメートル×10mmの培養皿中の種子細胞。 24時間後に約50％コンフルエンスを確実にするために必要な細胞の量を使用する。 注：我々の実験では、HT-1080細胞株およびプレート培養皿あたり1×10 5個の細胞を使用しています。 </li><li>キャリブレーションに使用する実験とプレート5追加の培養皿のために必要な培養皿の数を決定します。 </li><li>少なくとも24時間、37℃/ 5％CO 2インキュベーターに置き培養皿。 </li></ol> 3。のpHセンシングプローブの調製を<ol><li>のddH 2 0での蛍光pHプローブ8-ヒドロキシピレン-1,3,6トリスルホン酸三ナトリウム塩（HPTS /ピラニン）の1Mストック溶液を準備します。この溶液を室温で保存し、光から保護されなければならない。 </li><li> 1 mMの5のストック溶液を調製 - （および6） - カルボキシseminaphthorhodafluorアセトキシメチルエステル酢酸（C-SNARF-1 AM; therea呼ばれるFTER SNARF-1）のDMSO 1.76ミリリットルでSNARF-1 1mgを溶解することにより。徹底的に繰り返さピペッティングにより混和する。 SNARF-1溶液は-20℃で保存し、光から保護されなければならない。 </li></ol> 4。細胞標識<ol><li> 16時間生細胞イメージングの前に、完全培地の2ミリリットルを含む各培養皿に1M HPTS原液2μL（最終濃度1 mm）を追加します。 </li><li>インキュベーターに培養皿を返します。 HPTSプローブは、したがって、ラベルにプローブはピノサイトーシスによって取り込まれなければならない小器官、細胞非透過性である。この条件は、HPTSによる細胞のインキュベーションは、血清添加培地中で行われることを必要とする。 </li><li> HPTSとのインキュベーション後、滅菌PBSで細胞を2回洗浄し、少なくとも2時間、無血清培地2mlを加える。 SNARF-1とのインキュベーションは、血清は、プローブの取り込みを防ぐことができるため、無血清培地中で行う必要がある。 </li><li> AFを得るために、1 mMのSNARF-1のストック溶液10μlを追加inal 510μMの濃度と細胞は薄暗い光の下で、37℃で20分間インキュベートすることができます。 C-SNARF-1濃度およびインキュベーション時間は、細胞型に応じて変化し得る。 </li><li>インキュベーションの最後に、滅菌PBSで細胞を2回洗浄し、無血清培地2 mlを加える。 </li></ol> pHの調節因子の5。追加<ol><li>方法は、細胞内のpHの変化を検出することができるかどうかを評価するために、細胞内pHを調節する異なる試薬を使用することができる。 <ul><li>フィロマイシンA1、V型ATPaseの5の特異的阻害剤</li><li> + / H +交換アイソフォーム1（NHE-1）6 NA </li><li> NH 4 Clで、細胞内のpHが7を増加弱塩基</li></ul><ol><li>上記の試薬のストック溶液を準備します。 <ul><li>フィロマイシンA1：100μMの原液を得るために、DMSOを160μlにバフィロA1の10μgのを追加</li><li> EIPA：obtaiメタノールの1.67ミリリットルにEIPA 25mgを追加50 mMの最終濃度はna </li><li> NH 4 Cl を 1 mMの最終濃度を得るためのddH 2 O 10ml中の塩化アンモニウム0.53gを溶解する。 </li></ul> 注：すべての原液を-20℃で保存し、光から保護しなければならないフィロマイシンA1を除いて4℃に保たれている必要があります。 </li><li>後述するように別々の培養皿に、試薬の1つを追加します。 <ul><li> 100μMのフィロマイシンA1（最終濃度100 nm）で2μL </li><li> 50 mMのEIPA（最終濃度25μM）の1μL </li><li> 1M 塩化アンモニウム（最終濃度20 mM）を40μlの</li></ul></li><li> 37℃で30分間、細胞をインキュベート</li></ol></li><li>画像取得に進みます。 </li></ol> 6。生細胞イメージング顕微鏡の仕様および取り込み設定<ol><li>使用して生細胞イメージングを行う40Xの目的は、スペクトル反転走査型共焦点microscoに搭載された電動XYステージ、環境的に制御（温度、湿度、CO 2）ステージインキュベーターでガラス底培養皿を収容するチャンバを備えたPE。 </li><li>ここで使用した共焦点装置（オリンパスFV1000）が装備されている。 <ul><li>ダイオードレーザーは405nm </li><li> 40 mWのアルゴンレーザー（458 nmの、488 nmの、515 nm）を</li><li>ヘリウムネオンレーザー（543 nm）と</li><li>蛍10X目標、NA0.4 </li><li> LUCPLFLN 40X目標、NA0.60 </li><li> PLAPONSC PLAN APO 60X油目標、NA1.4 </li><li>ダイクロイックミラーDM 405/458/488/543 </li><li> SDM 560およびSDM 640ダイクロイックフィルター</li><li> BA655-755バリアフィルタ</li><li>ビームスプリッタBS20/80 </li><li>画像収集および分析のためのデータ解析ソフトウェア、 </li></ul></li><li>ステージのインキュベーターを37℃に温めており、顕微鏡が設定されると、以前に共焦点ステージイメージング室にHPTSとSNARF-1とインキュベートした培養皿を置く。 </li><li> A関心領域の位置を押え上げは、ソフト取得の設定は、図1を調整する。 <ul><li> 2仮想チャネルセットを設定します。 </li><li> 525 nmおよび570での543 nmおよび放射コレクションのSNARF-1の励起 - - 600 nmの505で405 nmおよび放射コレクションでHPTSの励起に対応している第一段階を開始する。 </li><li> 525 nmおよび640で543であり、発光のコレクションのSNARF-1の励起 - - 650 nmの505で458 nmおよび放射コレクションでHPTSの励起に対応している第二段階を開始します。 </li><li>共焦点開口は、デフォルトでは175程度に設定し、最適な絞り値を手動で300程度に設定される必要がある。 </li><li> 0.4ミクロンの厚さの連続水平光学切片（800×800ピクセル）を積層して画像を撮影。 </li></ul></li></ol>注：40Xの目的は、同時に複数のセルを観察することが有用であるとオルガネラの閲覧に適しています。しかしながら、空間分解能と信号強度があるベティrは、60X油対物レンズを用いて達成した。さらに、2つの色素の間には励起または発光のクロストークは存在しない。 pHのセンシングプローブの7。校正<ol><li>キャリブレーションの前に、以前にHPTSとSNARF-1とインキュベートした培養皿から培地を除去し、滅菌PBSで2回洗浄した後、5キャリブレーションソリューションの1の2ミリリットルを追加します。 </li><li>共焦点段階室に培養皿を置きます。 </li><li>以下の設定に応じてタイムラプス画像取得に進みます。 <ol><li>実験用の画像や間隔期間の回数を設定します。 注：私たちは通常、最大30分間の画像間の1分間のインターバルを使用しています。 </li><li>各画像の取得との間で閉じるようにシャッターをプログラムします。 </li></ol></li><li>表計算ソフトで分当たりの蛍光強度を記録し、蛍光強度が安定している時間を決定する。 20分 - 細胞内のpHは、15日以降は通常安定している。 </li><li> REPEATは、残りの各校正液のための新しい培養皿を用いて、7.4に7.1を繰り返します。 </li></ol> 8。データ収集<ol><li>キャリブレーションが行われた後、フィールド当たり15〜20個の細胞を含む4-5ランダムに選択されたフィールドからのサンプルデータを取得する。 2つの画像は、フィールドごとに得られる。HPTS標識（小胞）とSNARF-1ラベル（細胞質ゾル）のための第二のために1。 </li><li>バックグラウンド蛍光（細胞外領域）をデジタル両方のプローブの蛍光画像から減算される。それぞれの保存された画像からの標識小胞および細胞質ゾルは、バイナリマスクを使用して選択される。各pH感知プローブは、蛍光強度を数値スケール（0-4,095階調スケール）で測定される。細胞小器官または細胞質内のすべてのピクセルの平均強度が計算され、値は、蛍光比を計算するために使用される。 </li></ol> 9。データ解析<ol><li>様々なソフトウェアパッケージは、データ分析のために市販されている。 すなわちFLUOVIEWソフトウェア。 </li><li>スプレッドシートでは、レコードの値と次のように蛍光比を計算する：HPTSの場合には、R =（458nmの F / F 405 nm）を、SNARF-1の場合、R =（F 644ナノメートル / F 584 nmの ）。 </li><li>このようなグラフパッドプリズムなどの科学的なグラフ作成ソフトウェアを使用してデータをプロットします。プロットは、pH（X軸）の関数としての蛍光比（Y-軸）を描くべきである。カーブフィッティングは、非線形回帰を使用して達成される。 </li><li>ステップ8.2で説明したように、サンプルの蛍光強度を収集し、蛍光比を計算する。 pH値に比率を変換するために較正曲線を使用する。 </li></ol>

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No conflict of interest declared.

異なる細胞区画の生細胞イメージングで定量的なpH測定を正確に外部の課題に対する細胞応答におけるpH変化を測定するために必要とされる。しかしながら、残る主要なハードルの一つは、簡単かつ具体細胞区画を標的化することである。この点において、いくつかの研究は、エレクトロポレーションまたはマイクロインジェクション8-10によって細胞に導入する細胞内pH指示薬として?...

<table><tbody><tr><td>Solutions for calibration</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td></td></tr><tr><td>D-Glucose (dextrose)</td><td>Fischer</td><td>D16-1</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Fischer</td><td>M65-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Fischer</td><td>M-1612</td><td></td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Nigericin</td><td>Calbiochem</td><td>481990</td><td>Toxic by inhalation or in contact with skin</td></tr><tr><td colspan="4">Keep protected from the light at 4 &amp;deg;C</td></tr><tr><td>Culture dishes &amp;empty; 35mm</td><td>BD Biosciences</td><td>354456</td><td></td></tr><tr><td>pH-sensing probes</td><td></td><td></td><td></td></tr><tr><td>8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td><td>Life technologies</td><td>H-348</td><td>Keep away from the light at room temperature</td></tr><tr><td>5-(and-6)-Carboxy SNARF&amp;reg;-1, Acetoxymethyl Ester, Acetate</td><td>Life technologies</td><td>C-1271</td><td>Keep away from the light and humidity at -20 &amp;deg;C</td></tr><tr><td>Dimethyl Sufoxide (DMSO)</td><td>Fischer</td><td>BP231-1&amp;nbsp;</td><td>Harmful</td></tr><tr><td>Phosphate buffered saline (PBS) 100X</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Fischer</td><td>M-12445</td><td>20 g</td></tr><tr><td>Sodium phosphate monobasic</td><td>Fischer</td><td>S369-1</td><td>115 g</td></tr><tr><td>Potassium phosphate monobasic</td><td>J.T Baker</td><td>3246-01</td><td>20 g</td></tr><tr><td>Deionized water</td><td>N/A</td><td>N/A</td><td>Bring to 1 L</td></tr><tr><td>Autoclave and adjust pH to 7.4</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS) 1X</td><td></td><td></td><td></td></tr><tr><td>PBS 100X</td><td>N/A</td><td>N/A</td><td>50 ml</td></tr><tr><td>Sodium chloride</td><td>Fischer</td><td>L-11621</td><td>40.5 g</td></tr><tr><td>Deoinized water</td><td>N/A</td><td>N/A</td><td>Bring to 5 L</td></tr><tr><td>pH modulators</td><td></td><td></td><td></td></tr><tr><td>Bafilomycin A1</td><td>Sigma-Aldrich</td><td>B-1793</td><td></td></tr><tr><td>5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td><td>Sigma-Aldrich</td><td>A3085</td><td></td></tr><tr><td>Ammonium chloride</td><td>Fischer</td><td>A649-500</td><td></td></tr></tbody></table>

simultaneous ph measurement in endocytic and cytosolic compartments in living cells using confocal microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

細胞内のエンドソーム/リソソーム細胞区画の両方におけるpHの同時測定のために、レシオメトリック蛍光性pH感知プローブSNARF-1及びHPTSを用いた。 HPTSはエンドソーム/リソソーム区画のレシオメトリックpH測定を可能にするのに対し、SNARF-1は、細胞質ゾルコンパートメントに制限されています。 16時間HPTSとセル負荷は、エンドソーム/リソソーム4での選択的局在することができます。 ...

Watch this Scientific Journal Video about Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy at JoVE.com

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

共焦点顕微鏡を用いた生細胞におけるエンドサイトーシスおよび細胞質区画における同時pH測定

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

simultaneous-ph-measurement-endocytic-cytosolic-compartments-living

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K ビュー.  Université de Sherbrooke. Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells. 

ビデオ: Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

電気化学と蛍光顕微鏡を用いたプロトン ポンプ膜酵素の研究のための単一リポソーム測定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

生化学

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

レシオメトリック蛍光顕微鏡によってファゴソームのpHを測定します

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

免疫・感染学

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

レシオメトリックpH指示薬を使用して好中球ファゴソームと細胞質のイメージング

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

共焦点顕微鏡を用いたクロスキングダムバイオフィルムの細胞外pHのモニタリング

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液胞と細胞質のpHの測定<emインビボで&gt;</em&gt;酵母細胞懸濁液に

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

生物学

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

二重蛍光系比測定法を用いたマクロピノソームのpH、レドックス化学、分解能の測定

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

細胞内局在や人身売買を監視するpHluorinタグ付き受容体を利用します

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

共焦点顕微鏡を用いた生細胞におけるエンドサイトーシスおよび細胞質区画における同時pH測定

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さらに動画を探す

この動画の章

電気化学と蛍光顕微鏡を用いたプロトンポンプ膜酵素の研究のための単一リポソーム測定

In vitro Monitoring of Extracellular pH in Real-Time

レシオメトリック蛍光顕微鏡によってファゴソームのpHを測定します

レシオメトリックpH指示薬を使用して好中球ファゴソームと細胞質のイメージング

共焦点顕微鏡を用いたクロスキングダムバイオフィルムの細胞外pHのモニタリング

液胞と細胞質のpHの測定

二重蛍光系比測定法を用いたマクロピノソームのpH、レドックス化学、分解能の測定

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

細胞内局在や人身売買を監視するpHluorinタグ付き受容体を利用します

共焦点顕微鏡を用いた生細胞におけるエンドサイトーシスおよび細胞質区画における同時pH測定

要約

さらに動画を探す

この動画の章

電気化学と蛍光顕微鏡を用いたプロトン ポンプ膜酵素の研究のための単一リポソーム測定

In vitro Monitoring of Extracellular pH in Real-Time

レシオメトリック蛍光顕微鏡によってファゴソームのpHを測定します

レシオメトリックpH指示薬を使用して好中球ファゴソームと細胞質のイメージング

共焦点顕微鏡を用いたクロスキングダムバイオフィルムの細胞外pHのモニタリング

液胞と細胞質のpHの測定

二重蛍光系比測定法を用いたマクロピノソームのpH、レドックス化学、分解能の測定

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

蛍光顕微鏡による生細胞内のミトコンドリアのカルシウムとミトコンドリア膜電位の同時測定

細胞内局在や人身売買を監視するpHluorinタグ付き受容体を利用します

電気化学と蛍光顕微鏡を用いたプロトンポンプ膜酵素の研究のための単一リポソーム測定