この作品は、PMケインにNIH R01 GM50322によってサポートされていました。著者は、私たちの研究室のために、これらのプロトコルを扱うための博士Rajiniラオ、酵母pHluorinプラスミドを提供するとレシオメトリックpH測定に関するアドバイスジョンズホプキンス大学、博士とグロリアA.マルティネスムニョスに感謝します。

1。 BCECF-AMを用いたin vivo において、液胞のpHの測定<ol><li>一夜希望の媒体中で測定する酵母菌株を50mlの液体培養を育てる。目標は、中期対数期（OD600（600nmでの光学密度）が約0.8サスペンション用の測定）で細胞を有することである。 </li><li>遠心分離によりペレット酵母細胞。増殖培地、以前に秤量されたマイクロチューブへの転送0.6ミリリットルでペレットを再懸濁します。ペレット60秒間2,000 xgで遠心で再び細胞。として完全に可能な限り上清を除去した後、細胞ペレットを量る。このボリュームの培養物は、約200ミリグラムの細胞ペレットを与える、バッファ200μlを400μlの最終容量を得るために添加されるだろう。0.5グラム/ミリリットル（w / v）の最終濃度にペレットを再懸濁する。 </li><li> DMSOで準備12mMのストックからの50mMの最終濃度で細胞懸濁液にBCECF-AM追加。よく混ぜた後、株式会社30分間30℃で細胞をubate。ロッキングプラットフォームまたはローラードラム上。 </li><li>細胞がインキュベートされていますが、キャリブレーション·バッファを準備します。 、50mMのHEPES（4 - （2 - ヒドロキシエチル）-1 - ピペラジンエタンスルホン酸）、50mMのKClを、50mMのNaCl、0.2 M酢酸アンモニウム、 - 較正緩衝液は50mMのMES（（N-モルホリノ）エタンスルホン酸2）を含有10mMのアジ化ナトリウム、および10mM 2 - デオキシグルコース、及びpHに調整されては、NaOHまたはHClであなたの校正範囲の適切な値。 （警告：アジ化ナトリウムは非常に毒性があり、慎重に扱われるべきである）は、野生型細胞内のpHの測定のために、我々はpHが5.5から6.5に複数のキャリブレーション混合物を準備しますが、より多くのアルカリ性の液胞のpHを有することが期待変異のために（ VMA変異体）、我々は追加の、より高いpH緩衝剤を調製だろう。 </li><li>アリコート2 15 mlコニカルチューブに各pH値の校正バッファmlの、そして最後の110μMのモネンシンの濃度及び15μMニゲリシン、Rを与えることモネンシン（15 mMストック）とナイジェリシンを（2 mMストック）を追加espectively。ボルテックスミキサーでよく混ぜる。 （注意：モネンシンとナイジェリシン両方が有毒であり、慎重に処理する必要があります。） </li><li>マイクロ2,000 xgで30秒間遠心分離によりBCECF-AMのインキュベーション時間が終了すると、ペレット細胞懸濁液。細胞を再懸濁し、上記のように、グルコースと遠心分離機を欠く増殖培地1mlのです。一度この洗浄ステップを繰り返し、その後、グルコースなしで増殖培地200μlのペレットを再懸濁します。氷の上に置きます。 </li><li>上記調製した各pHの校正チューブに細胞懸濁液20μlを追加します。 30-60分間30℃でインキュベートする。ローラードラム上。 </li><li>交互に励起波長450 nmおよび490 nmの535 nmでの発光波長との両方で測定するための蛍光を設定します。 30℃に試料室の温度を設定するキュベット内の混合物を連続的に撹拌しながら、すべての測定を実行します。 </li><li>細胞の増殖培地のpHに応じてpHを5〜7に調整した1mMのMES（の1.96ミリリットルを追加するND実験計画）。キュベットに細胞懸濁液20μlを追加します。蛍光測定を開始します。我々は継続的な運動と異なる実験の時間のポイントデータの両方を収集します。連続した運動データのために、我々は5分間毎に6秒の測定を行い、その後の50mMの最終グルコース濃度にブドウ糖を追加し、5〜10分間の測定を継続する。単一の時点を測定のために、我々は一般的に1〜5分で測定を行う。キュベットへの細胞の添加後、運動の測定と同様にグルコースを追加し、他の測定5分かかる。グルコース添加後。これらの添加を変化させることができ、追加の成分（阻害剤等）を添加することもできる。 </li><li>実験測定が完了した後、30℃のインキュベーションからキャリブレーションチューブを取り外し、蛍光のキュベットにそれぞれのための全体の2 mlのボリュームを転送します。すべての5秒は、各サンプルのために30秒の合計にわたって同じ設定（1.8を参照）での蛍光を測定します。 </li><li>Microsoft Excelに蛍光データをエクスポートします。 （我々の蛍光は、これはExcelにタブ区切り形式およびインポートでテキストとしてデータのエクスポートが必要です。）各キャリブレーション混合物のために450 nmの490 nmでの蛍光の比率を計算することにより、検量線を取得します。蛍光比は、その後、検量線を得るためにpH値に対してプロットされている。 </li><li>蛍光比に実験データを変換し、標準曲線を用いてpH値を計算します。液胞pHを、時間に対してプロット（pH変化の動態）や各種条件（ 図1）に基づいて、または異なる変異株で比較したができる。 </li></ol> 2。酵母pHluorinを用いたin vivo で細胞質のpHの測定<ol><li>標準プロトコルによってプラスミド酵母pHluorinで所望の酵母株を形質転換し、補完最小培地欠けウラシル（SC-ウラシル）で形質転換体を選択する。 </li><li>中期対数期（ODにSC-ウラシルで形質転換細胞を50mlの液体培養を育てる<suロ&gt; 600 = 0.8以下）。 </li><li>液胞のpHが、一般的に細胞質pH測定、pHは6-8のための適切なpH範囲のバッファーで説明した校正標準を準備します。キャリブレーション·バッファのアリコート2ミリリットルは、いくつかの15ミリリットルコニカルチューブに所望のpHに調整した。 （1.4）上記のようにモネンシンとナイジェリシンを加え、よく混ぜる。 </li><li>上記のように遠心分離によって細胞を回収し、増殖培地600μlのでペレットを再懸濁します。 30秒間5,000 rpmで遠心分離することにより体重マイクロチューブ、ペレット細胞に移す。再びグルコース（SC-ウラシル、-グルコース）、細胞をペレット化することなく、増殖培地1mlにペレットを再懸濁し、繰り返す。最後の遠心分離の後、可能な限り徹底的に上清を除去し、細胞ペレットを量る。無グルコースとSC-ウラシルで0.5グラム/ mlの最終濃度に細胞を再懸濁します。 </li><li>キャリブレーション·バッファの各チューブに細胞懸濁液20μlを加え、ボルテックスミキサーでよく混ぜる。 3でインキュベート0℃で60分間回転ドラムロータ上のC。 </li><li>波長405および485 nmで励起し、508 nmでの発光波長蛍光を設定します。 BCECF測定のために上述したように、単一の時点または運動の測定およびグルコースの添加を進める。 </li><li>キャリブレーションサンプルの蛍光を測定し、BCECF（1.10から1.11を参照）として検量線を作成する。蛍光比対pH値のプロットは、ほとんどの細胞質pH測定液（pH 6.0〜8.0）9の範囲にわたって線形であるため、実験的な蛍光比の測定が容易にpHに変換される。 </li></ol>

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著者らは、開示する利害の衝突を持っていません。

我々は、pHホメオスタシスの態様の数に対処するために、これらのプロトコルを利用している。例えば、我々は、4,5野生型およびV-ATPアーゼ欠損変異体細胞の細胞質およびpH応答を比較した。我々はまた、グルコース、3〜胞のpH応答に改変された成長条件、特に外pHの影響を調べた。重要なことは、我々が観察応答が量的pH測定する他の方法と、プロトンポンプ活動の変化を説?...

pHの恒常性は、すべての生物1,2の動的かつ高度に調整されたプロセスである。生化学的プロセスは厳密にpH値によって規制されており、細胞内の環境が常駐酵素の最適な活性を可能にするために狭いpH範囲に同調される。しかし、細胞内pHの恒常性は、環境pHの急激な変化、代謝シフト、および特定のシグナル伝達経路によって挑戦することができます。また、細胞内のpHが重要なシグナルとしての地位を提供することができます。最後に、多くの細胞小器官は、周囲の細胞質は異なるとオルガネラ固有の機能に不可欠である内腔pH値を維持します。 酵母サッカロミセス·セレビシエ株高等真核生物2でpHの恒常性メカニズムの数。リソソーム/エンドサイトーシス経路の酸性オルガネラにおいて、pHは、主に多くのexchanと連携して作用する、高度に保存された液胞プロトンATPアーゼ転位（V-ATPアーゼ）によって制御されるpH勾配に依存ジェール。すべての真核細胞は、またプロトンエクスポートメカニズムを持っています。菌類や植物、形質膜の第二、別個のプロトンポンプ、PMA1、輸出プロトン代謝および細胞質のpHおよび細胞膜電位の主要な決定要因であると考えられている。 S.の遺伝的柔軟性出芽酵母とその商業的重要性は、それのpHホメオスタシス2を研究するための非常に興味深く、重要なモデルました。 オルガネラの酸性化の主な要因であることに加えて、V-ATPアーゼは、高度に規制された酵素であり、私たちの研究室では、V-ATPaseの調節のメカニズムを理解することに興味があります。この目標に向かって、私たちは、液胞と細胞質のpHの生体内 pH測定に使用してきました：1）変異の影響その妥協V-ATPアーゼ活性を調べるためにこのようなグルコース欠乏とreaddition、2などの変化外条件）への応答を監視するために、及び3）COORを探索するオルガネラと細胞膜プロトンポンプ3-5 dination。これらの実験は、酵母細胞での使用に適して堅牢なレシオメトリックpH指示薬の開発によって可能になった。 （ - （および6） -カルボキシフルオレセイン2&#39;7&#39;-ビス- （2 -カルボキシエチル）-5）、哺乳動物細胞において細胞質ゾルのpHを測定するために広く使用されている、酵母液胞に蓄積植物らは最初BCECFことを示した代わりに、細胞質ゾル6。 BCECFのローカライズにおけるこの差はおそらくBCECF-AM（BCECFのアセトキシメチルエステル）と液胞の保持6からアセトキシメチルエステルの開裂を担当する液胞の多くの加水分解酵素、に起因している。アリら 7は、さらに、BCECFを使用して、液胞pH測定を開発し、蛍光プレートリーダー形式にこれらの測定値を適合。プラスミド媒介Rを発現させることにより酵母の細胞質のpHを測定する手段としてブレットら導入された酵母pHluorinatiometric pH感受性酵母特異的プロモーター9の制御下にGFP 8。 BCECFおよび酵母pHluorin両方の励起スペクトルは、pHに敏感であるので、それらは単一の発光波長で測定された2つの励起波長での蛍光の割合でレシオメトリックpH指示薬として使用され、pHが8,10の尺度を提供する。これらの酵母液胞および細胞質ゾルのpHセンサは、シングルセルおよび人口ベースの測定のために使用されている。単一セルの測定は6,11蛍光顕微鏡と画像解析によって行われる。二つの波長における液胞または細胞質蛍光はセル毎に測定する。集団ベースの測定は、適切な蛍光機能を備えたマイクロプレートリーダーのいずれかで、または蛍光で実行されます。それは詐欺中グルコースなどのコンポーネントを追加するための簡単​​なアクセスを提供するため、我々は一般的に、蛍光で我々の測定を行っている連続運動測定。液胞および細胞質ゾルのpHの測定のための我々の現在の実験プロトコルは以下の通りです。両方も容易にマイクロプレートアッセイに適合される。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

液胞および細胞質ゾルのpHは非常酵母細胞で規定さおよび全体のpHホメオスタシスにおいて中心的な役割を占めるている。私たちは、液胞や細胞質に局在するpH感受性蛍光プローブを用いたin vivoでのpHのレシオメトリック測定のためのプロトコルを記述します。液胞pHは、そのアセトキシメチルエステルの形態で細胞に導入する際に、酵母液胞に局在BCECFを用いて測定される。細胞質ゾルのpHは、酵母プロモーターの制御下で発現pH感受性GFP、酵母pHluorinで測定される。蛍光中の酵母細胞懸濁液中の蛍光比の測定のための方法が記載されている。これらのプロトコルを介して、さまざまな条件の下で、または異なる酵母変異体のpHの単一時点の測定が比較され、経時的なpHの変化が監視されている。これらの方法は、高スループット実験のための蛍光プレートリーダーフォーマットに適応されている。他のAP上のレシオメトリックpH測定の利点現在使用され、潜在的な実験的な問題と解決策、およびこれらの技術の将来の使用のための見通しに近づくにつれても記載されている。

50mMのMESでpH5に緩衝、 図1には、リッチメディア（YEPD酵母エキスペプトン-デキストラン）で栽培された野生型酵母細胞上で得られる液胞pHのデータを提示します。培地のpHは、特に最少培地のために、一晩成長中に非常に劇的に変化することができるので、我々は、多くの場合、緩衝培地で細胞を増殖し、我々は増殖培地のpHは液胞のpH応答3に影響を与えることが?...

Watch this Scientific Journal Video about Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions at JoVE.com

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

液胞と細胞質のpHは生酵母で測定することができる（<em&gt; S.セレビ</em&gt;）特定の細胞区画に局在レシオメトリック蛍光色素を用いて細胞。我々は、酵母における液胞に局在BCECF-AM、および細胞質レシオメトリックpH感受性GFP（酵母pHluorin）と細胞質のpHと液胞のpHを測定するための手順について説明します。

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 ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

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

19.7K ビュー.  SUNY Upstate Medical University. 液胞と細胞質のpHは生酵母で測定することができる（ S.セレビ）特定の細胞区画に局在レシオメトリック蛍光色素を用いて細胞。我々は、酵母における液胞に局在BCECF-AM、および細胞質レシオメトリックpH感受性GFP（酵母pHluorin）と細胞質のpHと液胞のpHを測定するための手順について説明します。

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

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

生物学

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

シングルおよび多細胞コンテキストでInvadopodia媒介細胞外マトリックスタンパク分解の定量測定

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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.

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

スペクトラルフローサイトメトリーにより固形組織から単離された細胞懸濁液の分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母の4D顕微鏡検査

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

酵母におけるポリグルタミン毒性の試金を使用して融合パートナーの蛍光タンパク質の効果

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

そのまま線虫の酸素消費量の測定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...