The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

ここで説明するプロトコルは、急性リンパ芽球性白血病細胞株NALM6を使用するが、他の細胞型に適用することができます。 1.ソリューションおよび試薬<ol><li>完全RPMI <ol><li> 56ミリリットルのウシ胎児血清（FCS）およびRPMI-1640培地500mlボトルに5.5ミリリットルの200mMのL-グルタミンを追加します。 </li></ol></li><li> BrdUの原液<ol><li>ダルベッコのリン酸緩衝生理食塩水（DPBS）で32.5 mMのBrdUを（10mg / ml）を準備します。 </li></ol></li><li> BrdUを完全RPMI <ol><li>完全RPMIの10mlまでのBrdUストック溶液6.2μlを添加します。 </li></ol></li><li> DNase溶液<ol><li> 1mgのDNアーゼ/​​ DPBSでミリリットルを準備します。 </li></ol></li><li>染色緩衝液<ol><li> DPBSで3％の熱不活性化FCSおよび0.09％アジ化ナトリウムを準備します。 </li></ol></li><li>固定バッファ、透過処理用緩衝液および洗浄用緩衝液の定義については、物質一覧を参照してください。 </li></ol> 2.細胞いいえTE：細胞はより大きい6ヶ月間培養しませんでした。この方法は、細胞密度および培養培地の調整と非付着性細胞株に直接適用可能です。実験の開始時に指数関数的に増殖している細胞を使用してください。 <ol><li>完全RPMI中のT-75培養フラスコ中NALM6細胞を維持します。クラスII安全キャビネットを使用して、無菌条件下で、すべての手順を実行します。 <ol><li>週に3回文化を分割して、1ml当たり1〜2×10 6個の細胞間NALM6細胞を維持します。 </li><li>大気中のCO 2、5％、37℃でインキュベートします。 </li></ol></li></ol> BrdUで細胞の3パルス標識注意：それは潜在的な変異原と催奇形物質であるように注意したBrdUを処理します。 <ol><li> 5分間、150×gで遠心分離した細胞。注：新鮮な培地に細胞を転送すると、結果の再現性を向上させます。 </li><li> 2×10 6℃での完全RPMI中の細胞数および再懸濁細胞を行いますells / mlの。 </li><li> BrdUの完全RPMIに1×10 6細胞/ mlの最終細胞濃度を産生して細胞を2 1に希釈します。 </li><li>その後、完全RPMIで10のセル1を希釈し、45分間、5％CO 2で37℃でインキュベートします。遠心セル5分間150×gで、慎重に上清をすべて廃棄します。 </li><li>完全RPMIの少量（〜100μL）中で再懸濁した細胞は、細胞計数を行い、1×10 6細胞/ mlに調整します。 </li><li>ピペット48ウェルプレートのウェルへの細胞の1ミリリットル。ピペットより再現性のある結果を得るために、未使用のウェルにDPBSの1ミリリットル。 </li><li>所望ここ時点、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、空気中5％CO 2中で37℃でインキュベートします、18、19、20、21、22、23、及び24時間。注意：時間の長さは、実験計画を測定することを目的とするものに依存します。 </li><li>ピペットを用いてFACSチューブにすべてのセルを転送します。 1ミリリットルvoluとよく順次をすすぎます5ミリリットルの最終全容量のPBS MES。 </li><li> 5分間150×gで遠心分離し、慎重にすべての上清を除去します。細胞は、染色のための準備ができているすぐにこの（セクション4）を行います。 </li></ol> 4.細胞染色注：細胞表面染色が必要な場合は、細胞が全体に4℃に保たれることを確実に定着する前にそれを行います。 <ol><li>染色緩衝液100μl中に再懸濁細胞（任意の表面染色のためには、表面抗原と4℃で30分間インキュベートし、抗体の推奨量を追加します）。 </li><li> 、染色緩衝液1mlを加え150×gで5分間遠心分離し、上清を捨てます。 注記：特定の抗体濃度、インキュベーション時間等は、特定の実験の目的に応じて変化します。 </li><li>固定および透過処理<ol><li>固定緩衝液100μl中に細胞を再懸濁し、室温で15分間インキュベートします。 </li><li> 1ミリリットルOを追加F洗浄バッファー、150×gで5分間遠心分離し、上清を捨てます。 </li><li>透過化緩衝液100μlで細胞を再懸濁し、氷上で10分間細胞をインキュベートします。 </li><li> 、洗浄緩衝液1mlを加え150×gで5分間遠心分離し、上清を捨てます。 </li><li>チューブあたり固定緩衝液100μl中に細胞を再懸濁し、室温で5分間インキュベートします。 </li><li> 、洗浄緩衝液1mlを加え150×gで5分間遠心分離し、上清を捨てます。 注：必要に応じてプロトコルは、ここで一時停止することができます。染色緩衝液中に再懸濁させた場合に固定された細胞は、4℃で数日間安定です。先に進む前に、遠心分離後染色バッファーを削除します。 </li></ol></li><li> DNase処理<ol><li> DNase溶液100μl中に再懸濁細胞（30μgの6 DNアーゼ/ 10の細胞）および37℃で1時間、細胞を培養します。 </li><li> 、洗浄緩衝液1mlを加え5分間、150×gで遠心分離し、上清を捨てます。 </LI&gt; </ol></li><li>抗体染色 注：BrdUの以外の細胞内マーカーについて染色BrdU染色と同時に行うことができます。 <ol><li>重要：それぞれの単一の蛍光色素で標識された未染色の細胞と細胞か​​らなる補償コントロールを準備します。理想的には、実験チューブに使用されるもののような補償のコントロールに同じ抗体を使用しています。これが不可能な場合には、高度に発現される抗原に対する代替抗体は、同一の蛍光色素にコンジュゲート。 </li><li>洗浄緩衝液50μl中に細胞を再懸濁し、1μL/ BrdU抗体の10 6個の細胞を追加します。注：他の特定の細胞内抗原に直接結合した抗体を添加することもできます。 注：セリン10上でリン酸化ヒストンH3に対する抗体は、G2中の細胞を区別するために使用することができ、M、ヒストンH3のセリン10、有糸分裂の間にリン酸化されるTyr15がリン酸化CDC2に対する10抗体は、HAV細胞を検出するために使用することができます。E有糸分裂にコミット。11 </li><li>室温で20分間細胞をインキュベートします。 </li><li> 、洗浄緩衝液1mlを加え5分間、150×gで遠心分離細胞と上清を捨てます。 </li></ol></li></ol><ol start="6"><li>細胞周期分析のための染色DNA <ol><li>ペレットを緩め、7-AAD溶液（0.25μgの）の20μlを添加します。注：これは、7-AAD /セルの一定量を使用することが重要です。 </li><li>染色緩衝液1mlで細胞を再懸濁します。 </li></ol></li></ol>フローサイトメトリーデータの5集注：必要なマシンが使用する蛍光色素の数と性質に依存します。 <ol><li>リニアスケールのFSC-A、SSC-A、FSC-H（FSC-Wの代わりにFSC-Hを用いることができる）と7-AAD蛍光：次のパラメータを収集します。対数スケール上のAPCチャネルを収集します。対数スケールを用いて表面や内部のラベルの評価のために必要な追加のチャネルを収集します。 </li><li> COMを行いますサンプルを分析する前に、異なる蛍光色素との間で観察された発光スペクトルの信号の重複pensation。注：ほとんどのフローサイトメーターは、これを自動的に実行します。 </li><li>各サンプルの少なくとも10,000個のイベントを収集します。 </li></ol>フローサイトメトリーデータの分析6. 注：FlowJoソフトは、フローサイトメトリーデータ分析のために、本研究で使用したが、他のソフトウェアパッケージを使用することもできます。ゲーティング戦略は、図1に示されています。 <ol><li> FSC-AおよびSSC-Aのパラメータを使用して、生細胞集団を特定します。 </li><li>この集団内ダブレットと（もここで用いることができるFSC-W）FSC-AおよびFSC-Hを用いて凝集体を除外します。 </li><li>この集団内でのy軸上のx軸とのBrdU-APCに7-AADを用いたドットプロットを設定します。 </li></ol><img alt="図1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> 図1：ゲーティング戦略左PA。ネル：非ゲートの細胞は、SSCドットプロット対FCS-Aに示されています。生存可能な細胞集団を示したゲートによって識別されます。センターパネル：左パネルからゲート細胞は、FSC-Hドットプロット対FSC-A（FSC-Wの代わりに高さを使用することができます）に表示されます。ダブレットと凝集体が同定され、示されたゲートで除外されています。右パネル：中央のパネルで二重排除日からゲート細胞は、APC-ドットプロット対7-AADに表示されます。 BrdU抗体は、APCをパルス標識中のBrdUを取り込んだ細胞の同定を可能に標識されています。 7-AADは、DNA含有量についての情報を提供します。上部ゲートは、BrdUのための陽性細胞を定義するため、S期でのBrdUパルスの時に、左下のゲート、G 0/1のセルと右下のゲートGのもの2 / M。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">表示するにはこちらをクリックしてくださいこの図の拡大版。</a> <ol start="4"><li>細胞周期A<html> nalysis <ol><li>ダブレット排除ゲート内の細胞の最初のデータ·ファイルとゲートを開きます。 </li><li> （FloJoソフトウェアのプラットフォームの下に位置する）は、細胞周期分布のために、この集団を分析し、ディーン·ジェット·フォックスモデルを使用します。 </li><li>ゲートを作成し使用してG 0/1およびG 2 / Mピークの位置を取得します。 </li><li> BrdU陽性細胞と被写体上のゲートと同じ細胞周期分析にこの集団。 </li><li>作成ゲートから同じゲートを適用し、G 0/1およびG 2 / Mピークの位置について（作成したゲートを使用）制約を設定することにより、G 0/1およびG 2 / Mピークの位置を提供します。これは、 図2の第2のパネルに示されています。 注：他のソフトウェアは、データを分析するために使用されてもよく、命令は、それに応じて変化するであろう。 </li></ol></li></ol>840 / 52840fig2highres.jpg &quot;幅=&quot; 700 &quot;/&gt; 図2：細胞周期の進行最初のパネル（全細胞）を二重排除ゲートによって定義された細胞集団にゲートされます。この集団は、X軸上の7-AADとヒストグラムで表示しました。 G 0/1ピークのピークが軸の下の矢印で示されています。 図1に示すように、後続のパネルでのBrdU陽性細胞にゲートされている。得られたG 0/1位置の値を二重排除ゲートのゲートは、FlowJoソフト細胞周期ソフトウェア内のBrdU陽性のゲート細胞に適用した場合。 図1および第2のパネルに示すように、全体の人口を分析したときに得られる値に基づいて、G 0/1ピークの位置に示すように後続の各パネルには、BrdU陽性集団にゲートをしました。同じSAMPでのBrdU陽性細胞のためのG 0/1集団の位置を特定するためのBrdU陰性画分を使用しましたルは、サンプル間のDNA染色の強度のわずかな違いを制御します。各パネルに表示される数は、BrdUのパルスが終了してからの時間を表しています。計算された細胞周期相は日陰緑色で示されている。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+nuclear+and+cytoplasmic+cell+cycle+progression+by+drugs+employed+in+cell+synchronization.">Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization.</a> Experimental Cell Research. 219, 159-168 (1995).</li></ol>

The authors have nothing to disclose.

細胞周期を分析する能力は、癌生物学および細胞増殖と成長に影響を与える両薬物および遺伝子の作用機序を理解するために重要です。報告によると、細胞増殖を測定するアッセイの多数がありますが、大半は単に本数細胞を示す尺度を提供します。これらは、直接可視化、カウント、代謝活性またはATP濃度によって細胞数を測定するアッセイが含まれます。これらの方法の多くの主な利点?...

細胞周期の進行の間に細胞内で起こる細胞周期の機能と変更点の評価は、生物学の多くの側面、特に癌の生物学を理解するための基本です。悪性腫瘍の治療のために開発中の多​​くの薬剤は、細胞周期の進行に大きな影響を持っているか、細胞周期依存性の機構を介して、細胞死を誘導します。細胞周期の特定の期における細胞周期動態または細胞を研究するためには、細胞を同期させることが一般的です。しかし、同期化方法は、潜在的に得られた結果を混乱させる、研究された細胞に有害な影響を有し得る。1近年、細胞周期の特定の段階でのみ存在する蛍光タグ付きタンパク質の使用は、時間をかけて、単一細胞における細胞周期進行の分析を可能にしました2、ただし細胞は遺伝的にこれを読み取ることができるシステムへの使用を制限する、これらの標識タンパク質を発現するように操作する必要性を検討しますILY達成。 細胞周期は、2つのアクティブフェーズで構成されています細胞分裂が起こる合成DNAが複製される（S）相、有糸分裂（M）を。これらの相は3ギャップ相、G 0、G 1及びG 2で分離されています。 G 0または休止は、細胞は、細胞が前のDNA複製および細胞増殖は、DNA複製の完了との間が、細胞分裂の前に続けてG 2のサイズの増加ここで、G 1は 、周期を離れた休止期です。細胞周期の進行は、チェックポイントの数によって制御されます。環境条件がDNA合成の支援ではなく、S期への進入を防止するときG 1チェックポイントが活性化されます。イントラS期チェックポイントまたは遅延は、停止した複製フォークで生じ得るDNA損傷によって誘発することができます。 G 2の間、複製されたDNAの忠実性が確認され、損傷をG 2、検出された場合</サブ&gt;チェックポイントは、前の細胞分裂にDNA修復を可能にする活性化されます。有糸分裂の間の最終的なチェックポイントは、細胞分裂が正常に完了することができるように、染色分が正常に分裂プレートに整列されていることを保証します。これらのチェックポイントの3活性化は、一般的に細胞集団を同期するために使用されます。細胞周期チェックポイントは、多くの要因によって活性化することができるが、癌生物学において最も一般的には、DNA損傷の検出です。 DNA損傷応答はPI3キナーゼ様キナーゼ毛細血管拡張性運動失調およびRAD3関連（ATR）は、それぞれ、下流のエフェクターキナーゼのChk1およびChk2での活性化血管拡張性失調症変異した（ATM）によって開始される。3イベントの範囲は、ストールを含めたChk1を活性化させます複製フォーク、DNAの架橋、および紫外線損傷はChk2では、主に二本鎖切断によって活性化されます。 細胞周期iの長さに変更された条件の影響を研究するための通常の方法細胞周期の特定の段階において細胞を同期させるための1本は、いくつかの方法を介して達成することができます。細胞は、物理的サイズ、密度、側方散乱（粒度）、および細胞表面マーカーの発現に基づいて分離することができます。具体的には、細胞は、化学的手段によって同期させることができます。チミジン、ヒドロキシ尿素及びシトシンアラビノシドのようないくつかの薬剤は、薬剤が除去された後に循環を続けるS期における細胞の蓄積を生じ、細胞周期のS期におけるDNA合成を阻害するために使用することができます。またはM期のDNA含量-有糸分裂紡錘体、G 2と逮捕の形成を防止するノコダゾール、で処理した細胞。 G 0期の細胞の蓄積培地結果から血清の排除。培養血清中の栄養素の再添加は、細胞の正常な循環を再起動します。しかし、これらの同期方法の全ては、細胞の正常な循環と成長を妨害することが可能とresul有意な細胞死でT。 急性リンパ芽球性白血病細胞の同期は特に困難であり、これらの細胞は、遺伝子操作に適していません。ここで説明する方法は、細胞周期動態の評価と、従来の同期または遺伝子改変することなく、細胞周期の特定の段階にある細胞の研究を可能にします。この方法は、遺伝子改変し、従来の同期手順は容易に達成されていない他の細胞型のために有用であり得ます。この方法は、細胞の短期の成長および増殖にはほとんど影響を与えブロモデオキシウリジン（BrdUの）取り込み、長い確立使用に基づいている。4設立のBrdUプロトコルはS期の間に新たに合成されたDNAへのBrdUの取り込みを利用します。これは恒久的にBrdU露光中、S期にあったように、細胞をマークします。この集団は、BrdUのincorporために染色することにより、後の時点で識別することができエーション、それにより追跡し、細胞周期の通過に対する薬物効果の研究を可能にする時間をかけて評価することができる同期集団として機能します。 BrdUを通常DNアーゼまたは酸処理した後の達成、前抗体染色にさらされる必要がある。6,7取り込まれたBrdUは、追加のマーカーを含めることができ検出するために、フローサイトメトリーを使用しました。最も重要なのは、試験の開始時にS期にあった細胞の細胞周期分布の評価を可能にする、DNA含有量を測定するための色素の使用である。8さらに、追加の表面または細胞内抗原はまた、研究することができる。9、これらなどのKi67として、またはそのような切断されたカスパーゼ3のようなアポトーシスマーカーとしての一見無関係な細胞機能への細胞周期のイベントに関連してもよいです。潜在的な用途は、研究者の想像力によって制限されています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>APC BrdU Flow Kit</td>
			<td>BD Biosciences</td>
			<td>552598</td>
			<td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm 
			Buffer (referred to as Fixation Buffer)</td>
		</tr>
		<tr>
			<td>BD Cytoperm Permeabilization Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>561651</td>
			<td>Referred to as Permeabilization buffer</td>
		</tr>
		<tr>
			<td>BD Perm/Wash Buffer</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Referred to as Wash buffer</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>D-4513</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 12 x 75-mm FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR FORTESSA flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FORTESSA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman</td>
			<td>Gilson</td>
			<td>P2, P20, P100, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Gln 500 ml</td>
			<td>Lonza</td>
			<td>12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>FisherBiotec</td>
			<td>FBS-7100113</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine</td>
			<td>Sigma</td>
			<td>B5002-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon TC 150cm2 vented Flasks</td>
			<td>BD Biosciences</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25mL</td>
			<td>Greiner</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 200&#181;L</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 1mL</td>
			<td>Interpath</td>
			<td>24800</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Spintron</td>
			<td>GT-175R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Binder</td>
			<td>C 150</td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td>
			<td>Cell Signalling</td>
			<td>9708S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td>2197S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td><a name="RANGE!C23">2348S</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6&#160;</td>
			<td>DSMZ</td>
			<td>ACC-128</td>
			<td></td>
		</tr>
	</tbody>
</table>

temporal tracking of cell cycle progression using flow cytometry without the need for synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

この方法は、情報の範囲を得るために使用することができます。いくつかのアプリケーションは、ここで概説されています。 細胞周期の継続時間の評価細胞周期を介して通過する細胞のために必要な時間を決定するために、細胞を、BrdUのパルス後の種々の時点で採取します。評価の間隔は、分析される特定の細胞に適応させることができます。造血...

Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

同期を必要としないフローサイトメトリーを使用した細胞周期進行の時間的追跡 |生物学 |ビデオ

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

このプロトコルは、特定の時点でS期にあった細胞の時間的な追跡を可能にするために、ブロモデオキシウリジン（BrdUの）取り込みの使用が記載されています。 DNA色素と抗体標識の添加は、後の時点でS期細胞の運命の詳細な分析を容易にします。

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

temporal-tracking-cell-cycle-progression-using-flow-cytometry-without

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Biology

Cellular Processes

Cell Cycle and Division

SCIENTISTS IN ACTION

19.1K ビュー.  Westmead Millennium Institute for Medical Research and University of Sydney. このプロトコルは、特定の時点でS期にあった細胞の時間的な追跡を可能にするために、ブロモデオキシウリジン（BrdUの）取り込みの使用が記載されています。 DNA色素と抗体標識の添加は、後の時点でS期細胞の運命の詳細な分析を容易にします。 

ビデオ: Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

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 ...

生物学

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 ...

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 ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

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 ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

の同期<em&gt;カウロバクターCrescentus</em&gt;細菌細胞周期の調査のため

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

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.

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 ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

フローサイトメトリーを用いた組換えイオンチャネルの相対的な細胞表面および合計式の決意

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

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 ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...