Name: preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation
Uploaded: 2017-11-10T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

この作品は、NIH (TCC) に CA109821 によって支えられました。気前よく文書のコストをカバーするための資金を提供するためとセットアップと飛びだしシステムの運用に関する技術援助ベックマン ・ コールターに感謝します我々 は博士フレッド ・ ファルケンバーグが初期プライマリのすべての文化を提供することをありがちましょう。

注: このプロトコルは、B-すべての主要な細胞の直径は約 8 μ m (G1 期)、13 μ m (G2/M フェーズ) に範囲の最適化されています。したがって、特定の遠心速度とポンプ流量は異なるサイズの範囲のセルには適用できません。それにもかかわらず、沈降速度式を使用して適切な飛び出しパラメーターを推定できます。セル 1-3 10 6 セル/mL の x の最適密度で説明した 16 と...

<ol>
	<li>Beach, D., Piper, M., Shall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+newly-initiated+DNA+from+the+early+S+phase+of+the+synchronous+eukaryote,+Physarum+polycephalum.">Isolation of newly-initiated DNA from the early S phase of the synchronous eukaryote, Physarum polycephalum.</a> Exp cell res. 129, 211-221 (1980).</li><li>Sevcikova, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+cell+division+is+associated+with+maximum+telomerase+activity+in+naturally+synchronized+cultures+of+the+green+alga+Desmodesmus+quadricauda.">Completion of cell division is associated with maximum telomerase activity in naturally synchronized cultures of the green alga Desmodesmus quadricauda.</a> FEBS letters. 587, 743-748 (2013).</li><li>Rosner, M., Schipany, K., Hengstschlager, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+high-quality+biochemical+fractionation+with+a+refined+flow+cytometry+approach+to+monitor+nucleocytoplasmic+protein+expression+throughout+the+unperturbed+mammalian+cell+cycle.">Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.</a> Nat protoc. 8, 602-626 (2013).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+cell+synchronization.">Overview of cell synchronization.</a> Meth mol biol. 761, 1-23 (2011).</li><li>Langan, T. J., Rodgers, K. R., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Mammalian+Cell+Cultures+by+Serum+Deprivation.">Synchronization of Mammalian Cell Cultures by Serum Deprivation.</a> Meth mol biol. 1524, 97-105 (2017).</li><li>Dulla, K., Margalef, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Scale+Mitotic+Cell+Synchronization.">Large-Scale Mitotic Cell Synchronization.</a> Meth mol biol. 1524, 65-74 (2017).</li><li>Siefert, J. C., Clowdus, E. A., Sansam, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+in+the+early+embryonic+development+of+aquatic+animal+species.">Cell cycle control in the early embryonic development of aquatic animal species.</a> Comp biochem physiol Toxicol,pharmacol CBP. 178, 8-15 (2015).</li><li>Banfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+animal+cells+and+nuclei+by+centrifugal+elutriation.">Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation.</a> Nat protoc. 3, 663-673 (2008).</li><li>Grosse, J., Meier, K., Bauer, T. J., Eilles, C., Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+by+countercurrent+centrifugal+elutriation:+recent+developments.">Cell separation by countercurrent centrifugal elutriation: recent developments.</a> Prep biochem,biotechnol. 42, 217-233 (2012).</li><li>Smith, J., Manukyan, A., Hua, H., Dungrawala, H., Schneider, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Yeast.">Synchronization of Yeast.</a> Meth mol. 1524, 215-242 (2017).</li><li>Gascoigne, K. E., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+anti-mitotic+drugs+kill+cancer+cells?.">How do anti-mitotic drugs kill cancer cells?.</a> J cell sci. 122, 2579-2585 (2009).</li><li>Manchado, E., Guillamot, M., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+cells+by+targeting+mitosis.">Killing cells by targeting mitosis.</a> Cell death diff. 19, 369-377 (2012).</li><li>Komlodi-Pasztor, E., Sackett, D., Wilkerson, J., Fojo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+is+not+a+key+target+of+microtubule+agents+in+patient+tumors.">Mitosis is not a key target of microtubule agents in patient tumors.</a> Nature reviews. Clinic oncol. 8, 244-250 (2011).</li><li>Furst, R., Vollmar, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+perspective+on+old+drugs:+non-mitotic+actions+of+tubulin-binding+drugs+play+a+major+role+in+cancer+treatment.">A new perspective on old drugs: non-mitotic actions of tubulin-binding drugs play a major role in cancer treatment.</a> Die Pharmazie. 68, 478-483 (2013).</li><li>Kothari, A., Hittelman, W. N., Chambers, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Cycle-Dependent+Mechanisms+Underlie+Vincristine-Induced+Death+of+Primary+Acute+Lymphoblastic+Leukemia+Cells.">Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.</a> Cancer res. 76, 3553-3561 (2016).</li><li>Nijmeijer, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+primary+human+lymphoblastic+leukemia+cells+in+the+absence+of+serum+or+hematopoietic+growth+factors.">Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors.</a> Exp hematol. 37, 376-385 (2009).</li><li>Knudsen, E. S., Wang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+mechanisms+for+the+inhibition+of+E2F+binding+to+RB+by+cyclin-dependent+kinase-mediated+RB+phosphorylation.">Dual mechanisms for the inhibition of E2F binding to RB by cyclin-dependent kinase-mediated RB phosphorylation.</a> Mol Cell Bio. 17, 5771-5783 (1997).</li><li>Malumbres, M., Barbacid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cyclin-dependent+kinases.">Mammalian cyclin-dependent kinases.</a> Trends biochem sci. 30, 630-641 (2005).</li><li>Liang, H., Hittelman, W., Nagarajan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitous+expression+and+cell+cycle+regulation+of+the+protein+kinase+PIM-1.">Ubiquitous expression and cell cycle regulation of the protein kinase PIM-1.</a> Arch biochem biophys. 330, 259-265 (1996).</li><li>Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+gene+encoding+a+tRNA+synthetase-like+protein+is+enhanced+in+tumorigenic+human+myeloid+leukemia+cells+and+is+cell+cycle+stage-+and+differentiation-dependent.">Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent.</a> Proc Natl Acad Sci USA. 94, 6164-6169 (1997).</li><li>Ly, T., Endo, A., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+response+to+cell+cycle+arrests+in+human+myeloid+leukemia+cells.">Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.</a> eLife. 4, (2015).</li></ol>

CCE を使用して細胞周期の異なった段階でプライマリを取得するためのメソッドのすべてのセルを説明しました。最適な条件で G1 期の細胞の本質的に純粋な人口と G2/M の段階で細胞の高濃縮人口に容易に得ることができた優れた収率で、必要な場合、S 期で高濃縮のセルを取得することも。ここで示された結果は、独立した文化、すべて 215ですべての文化 (具体的には、すべ...

培養細胞は通常非同期的に成長し、個々 の細胞が細胞周期の異なった段階の存在。いくつかの生物は当然のことながら同期の細胞周期を展示または特定の生理学的な刺激によって同期することができます。たとえば、粘粘菌変形体の巨大なでん内核分割高同期ファッション1, と指数養魚は光と闇を交互に同期できる緑の藻の細胞期間2。このような生物は、ユニークな実験的属性を提供する間不十分な哺乳類細胞の複雑さをモデルします。人工的に哺乳類セル人口の同期機能細胞周期チェックポイントの分子基盤と細胞周期制御の理解を推進する中心となっています。血清剥奪、化学ブロック、リリース、または物理的特性3,4を利用する一般的な方法があります。血清の撤退、静穏化を入力するセルが生じる、再血清添加 G1 フェーズ5細胞周期再突入に昇格させます。阻害剤には、余分なチミジンやヒドロキシウレア G1/S 境界で細胞をブロック、M フェーズ3,4のセルを通常逮捕微小管阻害剤などのエージェントが含まれます。分裂振り払い間期細胞6より少ない付着しているので、分裂期細胞の豊かにするために使用できる物理特性を悪用する手法が含まれます。ただし、すべてのこれらの技術の潜在的な欠点があります。たとえば、すべてのセル型は分裂振り払いは有糸分裂阻害薬の事前同期せず限られた後血清や化学剤の存在の有無と細胞の収量で生存率を維持します。
初期胚細胞周期、場所細胞は分類7サイズが徐々 に減少を除いてほとんどの細胞周期を進める成長段階を経るし、サイズが大きくなります。このプロパティは、対向流遠心浄化 (CCE)、サイズの異なる細胞を分離する使用ことができますし、それゆえ細胞周期段階8,9の手法で利用されます。CCE、中に細胞が 2 つの反対勢力の影響の下で: 回転の軸から細胞を運転する遠心力と流体の速度 (対向)、回転 (図 1) の軸に向かってセルを駆動します。これらの力が等しい平衡位置を飛びだしチャンバーで細胞に到達します。平衡位置を指示、重要な要因は細胞径および密度です。緩衝液の率の増加の流れとして、対向ドラッグ力が遠心力を上回る新しい均衡を確立すると、商工会議所の内部のセルの位置が変更されています。すべてのセルが対向流率はその出口を促進するために十分に増加するまで商工会議所内より大きい細胞にとどまるに対し小さいものは最初、商工会議所のままで、その結果、チャンバー出口に向かってシフトされます。特定分数で対向速度の連続増加と飛びだし室をエスケープ細胞を集めることができるし、各分画に順番にサイズの増加のセルが含まれています。対向流率の増加と飛び出しを行う必要がなく遠心分離速度を減少させることによって遠心力の連続の減少は、同じ結果を達成するでしょう。浄化のプロセスには、セル型、採用、飛びだしバッファーおよび使用される特定の装置によって最適化が必要です。これらの条件下で細胞の堆積速度はストークスの法則によって最もよく述べ: SV = [d2(ρp- ρm)/18η] .ω2r、SV = 沈降速度;d = 粒子の直径Ρp = 粒子の密度Ρm = バッファーの密度Η = バッファーの粘度Ω =; ローターの角速度r = 粒子の半径方向の位置。したがって、SV は細胞径および密度に比例して、細胞周期を通って一般に定数である密度より大きく寄与直径が 2 番目の電源に発生するので。
CCE の重要な利点は、細胞が化学治療や栄養欠乏の過酷な条件にさらされないと本質的に動じない優れた収率で回収することができます。要件は、細胞周期にトラバース、問題の細胞が直径の少なくとも 30% の大幅な増加を受けることです。主な欠点は、特殊な遠心分離機、ローター、アクセサリーのコストです。それにもかかわらず、CCE によって豊かに細胞周期の特定の段階で細胞を準備する機能は、酵母から哺乳類セル9,10の生物で細胞周期研究を促進しています。また、最近の進歩で、細胞の分離の使用を拡大して癌組織、異なる種類の細胞、異種混合物でし免疫療法9の特定の細胞型の生産分離対健康からいます。
我々 の主要な関心のビンカ アルカロイドやタキサン系薬剤などのエージェント (Mta) 微小管作用のメカニズムを理解されています。これらの薬は、有糸分裂、スピンドル微小管機能11,12のブロックにのみ行動すると考えられていたが、最近の証拠は、彼らが間期の微小管13,14の対象にも示唆しています。我々 は最近、プライマリ急性リンパ芽球性白血病 (ALL) セル G1 期のいずれかの死を受けるまたは M 相ビンクリスチンと細胞周期依存的15の他の Mta で処理することを報告しました。CCE によって異なった細胞周期の段階にすべてのセルを分ける機能はこの結論に達するに尽力しました。この記事では、技術的な詳細を説明して、異なった細胞周期の段階ですべてのセルの CCE のプライマリの調製への応用のための実用的なヒントを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="1542">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Hanks&#39; balanced salt solution</td>
			<td style="width:108px;">Lonza</td>
			<td style="width:119px;">10-508Q</td>
			<td style="width:780px;">1 L bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Fetal Plus bovine serum</td>
			<td style="width:108px;">Atlas Biologicals</td>
			<td style="width:119px;">FP-0500-A</td>
			<td>500 mL bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">2-napthol-6,8-disulfonic acid dipotassium salt</td>
			<td style="width:108px;">Acros Organics</td>
			<td style="width:119px;">212-672-4</td>
			<td>100 g powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">50 mL concial tubes</td>
			<td style="width:108px;">Denville Scientifics</td>
			<td style="width:119px;">C1062-P</td>
			<td>500/case</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PES Membrane 0.22 &#181;m filter unit</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:119px;">SLGP033RB</td>
			<td>250/pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Avanti J-26S XPI w/ Elut. Non-IVD - 50/60 Hz, 200/208/240V</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">B14544</td>
			<td>centrifuge compatible with elutriation system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">JE 5.0 elutriator rotor kit</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356900</td>
			<td>rotor kit which includes the rotor, T-handle hex wrench, the quick release assembly (w/o the elutriation chamber), anchoring cable, and the strobe assembly</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chamber, standard, 4-mL, &#34;A&#34;</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">356943</td>
			<td>standard elutriation chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Easy-Load pump head</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07518-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex L/S Variable-Speed Drive</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07528-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Masterflex BioPharm platinum-cured silicone pump tubing, L/S 16</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">EW-96420-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">25 G x 1.5 Precision Glide needle</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">305127</td>
			<td>sterile, single-use needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">10 mL Luer-Lok syringe</td>
			<td style="width:108px;">BD</td>
			<td style="width:119px;">309604</td>
			<td>sterile, single-use syringes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">Vi-Cell XR</td>
			<td style="width:108px;">Beckman Coulter</td>
			<td style="width:119px;">383556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">PI/RNase staining buffer</td>
			<td style="width:108px;">BD Pharmingen</td>
			<td style="width:119px;">550825</td>
			<td>propidium iodide/RNase staining buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin B1 (GNS1) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">cyclin D1 (DCS-6) mouse monoclonal IgG antibody</td>
			<td style="width:108px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-20044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">P-Rb (S807/811) (D20B12) XPR rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">8516s</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:535px;">GAPDH (14C10) rabbit monoclonal antibody</td>
			<td style="width:108px;">Cell Signaling Technology</td>
			<td style="width:119px;">2118s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-mouse IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:535px;">Goat anti-rabbit IgG (H+L)-HRP conjugate</td>
			<td style="width:108px;">BioRad</td>
			<td style="width:119px;">170-6515</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of primary acute lymphoblastic leukemia cells in different cell cycle phases by centrifugal elutriation

セルを同期する機能を細胞周期の規則の私達の理解を進める中心となっています。一般的な技法は、血清遮断;化学物質の細胞を別の細胞周期段階で逮捕または分裂振り払い減少への遵守を悪用するの使用します。しかし、これらすべての欠点を持ちます。たとえば、血清飢餓動作も正常な細胞が、腫瘍細胞が癌遺伝子の活性化や腫瘍サプレッサーの損失のための危険にさらされた細胞周期チェックポイントのあまり。同様に、化学的に扱われる細胞集団は薬害被害を抱くし、ストレス関連の変更を表示できます。これらの問題を回避する手法が対向流遠心浄化 (CCE) つの反対勢力、遠心力と流体の速度、サイズおよび密度に基づいて細胞の分離の結果をセルを受けます。通常サイクルを進める細胞が拡大するので、細胞を別の細胞周期の段階に分割する CCE を使用できます。ここで我々 は急性リンパ性白血病の主にこの手法を適用します。最適な条件下で、優れた収率で、G1 期の細胞の本質的に純粋な人口と G2/M の段階で高濃縮細胞集団が取得できます。これらの細胞は、抗がん剤の作用の勉強の細胞周期依存性のメカニズムおよび他のアプリケーションには最適です。またどのように標準的な手順に変更の最適なパフォーマンスの結果を表示し、技術の制限について説明。紹介する詳細な方法は、アプリケーションや他の種類の細胞に手法の探査を促進するべき。

プライマリのすべてのセル (10 x 3 48) 遠心浄化を受けるし、プロトコルで説明されているように 2 つの洗浄分数と 20 の主な分数に収集されました。対応するローターの速度と同様、各画分のセルの合計数が表示されます代表的なデータを表 1 に示します。全体収率は通常 80% では以上。留分の細胞径の測定は、そのセル径に伴って飛び出し (図 2...

Watch this Scientific Journal Video about Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation at JoVE.com

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

このプロトコルでは、異なる細胞周期の段階に主な急性リンパ芽球性白血病細胞を分離する遠心浄化の使用について説明します。

遠心浄化によって異なった細胞周期の段階で主に急性リンパ性白血病細胞の調製

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

The overall goal of this procedure, is to isolate cells into different cell cycle phases using centrifugal elutriation. This method can help answer key questions in the cancer therapeutics field. Because the isolation of cells into separate phases, allows for the interrogation of phase dependent affects of anti-cancer compounds.The main advantage of this technique is that it allows for separation of cells into specific phases using physical properties, rather than chemical perturbations. To start the experiment, install the strobe assembly into the centrifuge such that the power cord is fed out of the chamber through the port on the left. Adjust the strobe flash lamp so that it lines up with the viewing window, when the centrifuge is closed.Secure the assembly in place, by tightening two phillips head screws, at the top of each bracket. And then by tightening the thumb screws at the bottom of the bracket. Plug the power cord into the strobe power port, located at the back of the centrifuge.Next, set the rotors straight down onto the centrifuge drive hub, and firmly secure it with a T handle hex wrench. Then, place the assembly containing the elutriation chamber, counterbalance, rotating seal assembly, and a mounting plate onto the rotor. Push down evenly, placing one hand on the elutriation chamber, and the other hand on the counterbalance, until the bolts on the rotor snap into place.Place the pin, located at one end of the cable, into the hole in the rotating seal assembly. And secure the eyehole, at the other end, with one of the phillips head screws, affixed onto the bracket to connect the anchoring cable. To assemble the flow system, to pump buffer into and out of the elutriation chamber, connect size 16 tubing to a variable speed pump.Insert one free end of this tubing through a port at the top left of the centrifuge chamber, such that it enters into the chamber. Attach a fitting to the free end of this tube, and then insert the fitting into the input hole of the rotating seal assembly. Lastly, insert the output tubing, through a port, to attach it to the transfer tube at the top of the rotating seal assembly.Once the elutriation system is assembled, sterilize it by placing the other free end of the input tubing into a 500 milliliter bottle, filled with 5%bleach. And placing the output tubing into an empty one liter waste bottle. Next, turn on the pump, and allow the bleach to flow all the way through the elutriation system, until it is pumped out into the waste bottle.Now, turn the centrifuge on and allow it to come to the maximum designated speed to release bubbles and back pressure. Once the set speed is reached, stop the centrifuge. After the rotor has stopped completely, open the lid and inspect the chamber for any leaks.Next, in a similar fashion, as previously described, run sterile water through the system for 10 minutes. After flushing with water, replace the water reservoir with a sterile glass bottle filled with 100 milliliters of elutriation buffer. Place the input tubing into this bottle, so that it is at the very bottom.Start the centrifuge, and allow the buffer to flow through the system. After the reservoir bottle is close to empty, and water is fully flushed out of the system, move the output tubing from the waste bottle into the reservoir so that buffer is continuously recycled through the system. Finally, switch on the strobe lamp, and turn the elutriation knob to the extreme left.Now, adjust the viewing window by slowly turning the knob to the right, so that the elutriation chamber is clearly seen, and the strobe light is in sync with the rotor's speed. Prepare the cell sample as described in the text protocol. Add the resuspended cells into the reservoir bottle containing the recycling elutriation buffer.Allow the cells to enter into the system and reach equilibrium within the elutriation chamber. To ensure that all cells are retained in the chamber, take a drop of the eluent on a glass slide, and view it under a microscope. If the sample is devoid of intact cells, it confirms the retention in the chamber.Once all the cells have entered into the chamber, and a distinct boundary at the whitest part of the chamber, containing cells and equilibrium is seen, begin collecting fractions. Move the output tubing to a pre-chilled 50 milliliter conical tube, labeled W1.Collect 50 milliliters of elutriation buffer in this tube and store on ice. Soon after collecting the W1 fraction, lower the centrifuge speed to 821 G.And move the output tube to the conical tube, labeled W2, and collect 50 milliliters more at this speed.Continue changing the speed, and collecting fractions. Make sure there is medium in the reservoir tank, and always store the collected fractions on ice. After all fractions have been collected, stop the centrifuge, and collect 50 milliliters in a conical tube, labeled stop.Pellet cells collected in each of the fractions, and using downstream analysis, such as flow cytometry, or western blotting, as required. A line graph displaying the average diameter of cells in successive elutriation fractions, is shown. Note that the larger the cell diameter, the higher the fraction number at which it elutes.Results from fluorescents activated cells sorting analysis, show the DNA content of cells in each of the 20 elutriation fractions. Early fractions, F1 to F5, contain almost exclusively cells with 2N DNA content representing cells in G1 phase. A mixture of cells in G1 and S phases, was seen in intermediate fractions, F6 to F9.And cells with 4N DNA content, representing cells in G2 to M, were observed starting in F10.Shown here, are western blot results from probing the levels of cell cycle related proteins in pooled fractions of cells in G1 phase, and G2 to M phase. The G2 to M Pool had higher levels of Phospho-Rb and Cyclin B one, compared to the G1 Pool, as expected. Cyclin D one signal was weak, but nonetheless was detectable in the G1 Pool, and undetectable in the G2 to M Pool.Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it is important to remember to take your time, and make sure that the elutriation system is assembled properly, and that all materials and solutions are prepared. Following this procedure, other methods such as cell cycle analysis, cell viability assays and immunoblots can be performed in order to interrogate how therapeutic compounds may be acting in a cell cycle phase dependent manner.

Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

サンプルおよび測定可能な残存病変と急性骨髄性白血病の白血病幹細胞を測定するプロセス骨髄に対する包括的なプロトコル

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

がん研究

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

フローサイトメトリーを診断とフォローで主に急性骨髄性白血病、白血病幹細胞患者由来異種での見積もりに

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

世代と複雑な染色体と細胞周期逮捕細胞の分離

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

主な白血病細胞の代謝プロファイルの評価

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

マウス造血幹細胞および前駆細胞およびMLL-AF9駆動白血病におけるミトコンドリア活性酸素種のフローサイトメトリック解析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

免疫蛍光による非小細胞肺癌患者からの循環腫瘍細胞の半自動PD-L1特性図と列挙

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

フローサイトメトリーによる無毒ストレス後のγH2AXとアポトーシスの細胞周期特異的測定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

核/細胞質タンパク質の密売を監視する原発性慢性リンパ球性白血病細胞の細胞分画

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

急性骨髄性白血病におけるバルク亜集団と幹細胞を区別するNKG2Dリガンド表面検出の2つのフローサイトメトリックアプローチ

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...