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>

<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 border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Solutions for calibration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">D-Glucose (dextrose)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">D16-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Magnesium sulfate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M65-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">HEPES</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Calcium chloride dihydrate</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-1612</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Nigericin</td>
			<td style="width:136px;">Calbiochem</td>
			<td align="right" style="width:119px;">481990</td>
			<td>Toxic by inhalation or in contact with skin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;">Keep protected from the light at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Culture dishes &#8709; 35mm</td>
			<td style="width:136px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH-sensing probes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS)</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">H-348</td>
			<td>Keep away from the light at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">5-(and-6)-Carboxy SNARF&#174;-1, Acetoxymethyl Ester, Acetate</td>
			<td style="width:136px;">Life technologies</td>
			<td style="width:119px;">C-1271</td>
			<td>Keep away from the light and humidity at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Dimethyl Sufoxide (DMSO)</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">BP231-1&#160;</td>
			<td>Harmful</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 100X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">M-12445</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium phosphate monobasic</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">S369-1</td>
			<td>115 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Potassium phosphate monobasic</td>
			<td style="width:136px;">J.T Baker</td>
			<td style="width:119px;">3246-01</td>
			<td>20 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deionized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Autoclave and adjust pH to 7.4</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">Phosphate buffered saline (PBS) 1X</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">PBS 100X</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>50 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Sodium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">L-11621</td>
			<td>40,5 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Deoinized water</td>
			<td style="width:136px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Bring to 5 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;"></td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:872px;">pH modulators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">&#160;Bafilomycin A1</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">B-1793</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-(N-Ethyl-N-isopropyl)amiloride (EIPA)</td>
			<td style="width:136px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Ammonium chloride</td>
			<td style="width:136px;">Fischer</td>
			<td style="width:119px;">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

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

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D共焦点顕微鏡を用いたゼブラフィッシュにおける顔面形態形成を分析する

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

フローサイトメトリーによる腎上皮細胞における細胞質ゾルカルシウム測定

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

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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