We thank J. Schmidt from the University of Freiburg for constructing the device. We thank P. Nielsen and D. Medgyesi for their support and critical reading of this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft through SFB746 and by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1.デバイスの設計と構築<ol><li> パイロット実験 注：特定の光遺伝学ツールおよび細胞型に必要な光強度が大幅に異なる場合があります。プロトタイプとパイロット実験は必要最小限の光強度を推定するのに有用です。以下の実験のために使用される光遺伝学ツールは、ドロンパ - リンカー - ドロンパ融合構築物です。ドロンパシーケンスは、商業的に（材料のリストを参照してください）を得ました。長いリンカーは、発現構築物は、分子内（分子間）二量体を形成することを可能にするために2つのドロンパ配列の間でクローニングしました。以下に説明する手順は、他の多くの光遺伝学ツールまたは光学的に制御された物質に適合させることができます。 <ol><li>パッケージング細胞からのレトロウイルス含有上清を用いて、形質導入のための製造元の指示に従ってドロンパ - リンカー - ドロンパ含む構築物でラモスBリンパ球を伝達します。事前に応じて細胞を収穫し、カウントviouslyプロトコル13を発表しました 。 </li><li>濃度で再懸濁し、細胞1×10 6培地中/ mLの（RPMI 1640、10％の熱不活性化FCS、2mM L-グルタミン、100U / mLのペニシリン、100 U / mLのストレプトマイシン、および50μMの2-メルカプトエタノール）ガラスFACSチューブに移します。 </li><li>フローサイトメーターの中にチューブを挿入し、ドロンパ（GFPチャンネル）14,15の蛍光強度を測定します。 </li><li>おそらく有害な波長から目を保護するために、すべてのさらなるステップのための保護メガネを着用してください。 </li><li>手動でチューブの外側に500 nmのLEDライトを配置し、ドロンパの蛍光強度を測定し続けます。 </li><li>ドロンパの蛍光強度が減少するまでのLEDライトの数および/または強度を増加させます。 </li><li>手動でチューブの外側に400nmのLEDライトを配置し、ドロンパの蛍光強度を測定し続けます。 </li><li>ドロンパのFLUOまでLED照明の数および/または強度を上げます強度が増加するrescence。 </li><li>ステップ1.1.5と1.1.7でphotoswitchingから必要であると予測されるようにデバイスを構築するために、少なくともとして多くのLEDライトを使用してください。 </li></ol></li><li> デバイスの構築 注：デバイスはフライブルク大学からプロの加工ショップ、Arbeitsgruppe Technikでによって建てられました。ほとんどの部分はカスタムメイドであり、市販されていません。各部分の正確な寸法は、 図1および図 2に示されています。この装置の組み立てを明確にするために、補足ビデオ1が設けられており、最も重要なステップは、以下に記載されています。 <ol><li> 図1Aに示されている正確な測定値を有するポリ塩化ビニル（PVC）からミル片A。 </li><li>ピースAのドリル穴にLEDライトを挿入し、水漏れを防ぐために、塩ビ接着剤で各穴を塞ぎます。 </li><li> （各波長が別々のchannを占有しなければならないマルチチャンネル変圧器にLEDを接続します0〜29ミリアンペアから調整可能な出力電力とエル）。 </li><li>物理的な損傷からLEDを保護するために描かれているようにピースAに3本のネジで、外側シース（B）を取り付けます。 </li><li>ピースAの開けた穴にチューブクリップ（C）内の水ポンプ、接着剤にデバイスを接続できるようにするには（ 図1aに示された、断面Y：9ミリメートルと61.5ミリメートル）のPVC接着剤を使用しました。 </li><li> 58.5ミリメートル長いプレキシグラス管（D1）をカットします。 </li><li> 図2に示すようにプレキシグラスから作品D2とD3を製造しています。 </li><li>プレキシガラス接着剤でD1の底に接着剤ピースD3。 </li><li>ピースD2にゴム製のOリングを取り付け、プレキシグラスの接着剤でD1の一番上に接着。 注：ピースD1、D2、D3が一緒にピースDを形成します</li><li> 3本のネジでピースAにピースDを挿入します。 </li><li>変圧器に電源を適用する前に水漏れのためのデバイスをテストします。 </li></ol></li></ol> 2.光遺伝学ツールの速度論を測定<ol&gt; <li> 細胞を準備 <ol><li> RPMI培地中で培養ラモスB細胞（RPMI 1640、10％熱不活性化FCS、2mM L-グルタミン、100U / mLのペニシリン、100 U / mLのストレプトマイシン、および50μMの2-メルカプトエタノール）3-密度10×10 6細胞/ mL、37℃で、5％CO 2。 </li><li> 5分間350×gで4℃での遠心分離により収穫ラモスB細胞。 </li><li>製造者の指示に従ってノイバウアー計数チャンバーを用いて細胞を計数し、600μL培地に1-5×10 6個の細胞を懸濁します。 </li><li>ガラスFACSチューブに細胞を移し、氷の上に置くと、測定まで、光から保護。 </li></ol></li><li> フローサイトメーターおよび機器を準備します。 <ol><li>加熱された水ポンプにデバイスを接続し、37℃に温度を調整します。 </li><li>ガラスFACSチューブを使用する場合は、ゴム製のOリングを用いて、フローサイトメーターの黒いOリングを交換し、さらにシリコングラムを適用します楽。 </li></ol></li><li> 測定 <ol><li>慎重にデバイスにガラスFACSチューブを挿入します （ 図3）フローサイトメーターに接続します。 </li><li>測定を開始し、ドロンパ蛍光強度（GFPチャネル）を記録。 </li><li>それぞれが400nmまたは500 nmの光で細胞試料を照射し、ドロンパの蛍光強度（GFPチャネル）を記録。 </li></ol></li><li> データ分析 <ol><li>ソフトウェアサイトメーターの適切な流れに実験データを分析します。側方散乱およびゲート生きた細胞に対してプロット前方散乱。経時ドロンパの蛍光強度をプロットします。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

LEDサーモフローは、フローサイトメーターで光遺伝学ツールを研究するための革新的なデバイスです。 これまでのところ、光遺伝学のサンプルを顕微鏡レーザーや懐中電灯のデバイス11,12のみで照らされてきました。試料に懐中電灯の角度と距離に応じて、照明の量の大幅な変動が実験間で期待されています。また、1人での実験で動作することができる懐中電?...

それらは経路1-4シグナルの配線を解読するために使用することができるので、光遺伝学ツールは、部分的に、人気を集めています。これらは、光を照射されたとき、それらの立体配座および結合親和性を変更するための光活性化タンパク質の能力に基づいています。シグナリング要素にこれらのタンパク質を融合して、複雑な細胞内シグナル伝達経路5-12内のシングルプレイヤーの特定の調節を可能にします。従って、シグナル伝達経路は、高い時間および空間分解能を用いて研究することができます。 ほとんどの細胞ベースの光遺伝学の研究では、生化学分析11,12に続く光の存在下での培養と組み合わせた顕微鏡ベースの方法を利用します。対照的に、フローサイトメーター、キャピラリーに沿って細胞をsingularizesセルサイズ、粒度と蛍光強度を測定します。この方法は、thousan分析する能力を含む、顕微鏡または生化学的方法を超える大きな利点を有しており、非常に短い時間で単細胞解像度で生細胞のDS。したがって、フローサイトメトリーを用いて光遺伝学を組み合わせることが望ましいです。 我々の知る限りでは、光遺伝学、フローサイトメトリーのための確立されたプロトコルはありません。広く受け入れられた手順は、手動で懐中電灯のデバイスとの反応管の外側から細胞を照射することです。しかし、フローのマニュアル照明は、サイトメーターの反応管を通過する光を必要とし、生細胞イメージング、円筒形、加熱された水室のため。これは、実質的な光散乱および光の損失を引き起こします。また、手動の照明によって提供される光強度は（ 等角度、距離、）実験間の再現性がないと一つの実験の波長の数には限界があります。 LEDサーモフロー装置を構築することによって、我々は、これらの制限を克服することができました。このデバイスでは、細胞は一時中で特定の波長で照射することができますフローサイトメトリー測定中erature制御方法。これは、実験内との間の光の正確で再現性の量を可能にします。 インビボでの我々の装置の有用性を実証するために、我々はphotoswitching中ラモスB細胞におけるドロンパの蛍光シグナルを記録しました。ラモスB細胞は、ヒトバーキットリンパ腫に由来します。ドロンパは、モノマー、ダイマーまたは四量体として存在する蛍光タンパク質です。そのモノマー形態では、非蛍光性です。 400 nmの光で照明は二量体化および四を誘導し、ドロンパタンパク質の蛍光をレンダリングします。このプロセスは、500 nmの光を照射することによって逆転させることができます。ドロンパタンパク質は、タンパク質4,13のシグナリング機能および位置を制御するために使用されています。 ここでは、フローサイトメーターでドロンパのphotoswitchingを研究するためにラモスB細胞にドロンパ - リンカー - ドロンパのタンパク質を発現しました。私たちのデバイスを使用して、我々は効率的にすることができたとリアルタイムでの蛍光強度を記録しながら再現性ドロンパをフォトスイッチ。この方法は、マニュアル照明で現在の照明プロトコルを介して実質的な利点を提供し、大幅に光遺伝学ツールとケージ化合物のための実験的なレパートリーを広げます。大幅に簡素化し、新たな光遺伝学ツールの発見と開発を加速する私たちのデバイスを使用しました。

<table border="0" cellpadding="0" cellspacing="0" width="1212">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Glass FACS tube</td>
			<td style="width:357px;">&#160;Thermo Fisher Scientific; Waltham, USA</td>
			<td style="width:312px;">14-961-26</td>
			<td style="width:296px;">Borosilicate glass tubes 12x75 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Flow Cytometer</td>
			<td style="width:357px;">BD Bioscienceg; Heidelberg, Germany</td>
			<td style="width:312px;">Fortessa II</td>
			<td>Special Order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dronpa: pcDNA3-mNeptune2-N</td>
			<td style="width:357px;">Addgene; Cambridge, USA</td>
			<td style="width:312px;">41645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PolyJet</td>
			<td style="width:357px;">SignaGen, Rockville, USA</td>
			<td style="width:312px;">SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 505 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">HLMP-CE34-Y1CDD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LED 400 nm</td>
			<td style="width:357px;">Avago Technologies; Boeblingen, Germany</td>
			<td style="width:312px;">UV5TZ-400-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas tube 15 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">76999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 3 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 2,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Plexiglas 1,5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">692215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 5 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC tile 6 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PVC block 50 mm</td>
			<td style="width:357px;">Maertin; Freiburg, Germany</td>
			<td style="width:312px;">690020.050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-Mercaptoethanol</td>
			<td style="width:357px;">EMD; Germany</td>
			<td style="width:312px;">805740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">FCS</td>
			<td style="width:357px;">PAN Biotech; Aidenbach, Germany</td>
			<td style="width:312px;">P30-3302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Strptomycin (10000 U/ml)</td>
			<td style="width:357px;">Invitrogen, Life Technologies; Darmstadt, Germany</td>
			<td style="width:312px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrifix plexiglas glue</td>
			<td style="width:357px;">Evonic industries, Essen, Germany</td>
			<td style="width:312px;">1R0192</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tangit PVC-U glue</td>
			<td style="width:357px;">Henkel, D&#252;sseldorf, Germany</td>
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		</tr>
	</tbody>
</table>

led thermo flow &#8212; combining optogenetics with flow cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

フローサイトメーターとLEDサーモフローを使用して装置の機能コアは光が内側に向いて円形に配置されたLEDれた円筒状のチャンバです。このチャンバーは給水ポンプ、LEDの温度を制御することができ、ならびに細胞サンプルに接続することができます。 LEDは、変圧器に接続され、したがって、各波長に対する光強度を個別?...

Watch this Scientific Journal Video about LED Thermo Flow Ã¢â‚¬â€ Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LEDサーモフロー - フローサイトメトリーで光遺伝学を組み合わせます

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

led-thermo-flow-combining-optogenetics-with-flow-cytometry

Research

JoVE Journal

Biology

8.5K ビュー.  Max-Planck Institute for Immunobiology und Epigenetics. Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

ビデオ: LED Thermo Flow — Combining Optogenetics with Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM：フローサイトメトリーにより検出免疫沈降

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

生物学

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

マウス減数分裂細胞のフローサイトメトリーの精製

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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 ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

フローサイトメトリーを用いて細胞外小胞の分析のための技術

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

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 ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

孤立した塊茎プロトプ ラストのフローサイトメトリーによる表現を測定

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

フローサイトメトリーによる細胞性粘菌キイロタマホコリカビマクロピノサイトーシスの高スループット測定

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...