Name: sample preparation for mass cytometry analysis
Uploaded: 2017-04-29T13:00:00.000+00:00
Duration: 6 min 28 s
Description: Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Work in the Barton lab was supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT RP110471). This research was supported in part, by a training grant fellowship for Ryan L. McCarthy from the National Institutes of Health Training Program in Molecular Genetics 5 T32 CA009299. The core facility that maintains and runs the mass cytometry machine is supported by CPRIT grant (RP121010) and NIH core grant (CA016672).

1.细胞收获<ol><li> 从原代组织的细胞收获 注意:从原代组织获取细胞所描述的方法是特别适用于小鼠乳房肿瘤组织和是从其他来源初生组织可能不适用。 <ol><li>通过将5mg的透明质酸酶和在每克组织10毫升DMEM / F12培养基中30毫克制备胶原酶消化缓冲液进行处理。过滤消毒消化缓冲液。 </li><li>隔离小鼠并记录肿瘤的重量初级乳腺肿瘤。 </li><li>讳言与＃10解剖刀组织进行至少5分钟。 </li><li>切碎的组织加至消化缓冲液（每克组织10毫升缓冲液中）的适当体积。 </li><li>在37℃下摇在消化缓冲液切碎的组织在100rpm 1-3小时。 </li><li>通过使细胞通过0.4微米的过滤器到50ml管获得单细胞悬液。 </li><li>离心细胞，在300×g下在4℃10分钟;C。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>在4mL DMEM / F12重悬沉淀，并转移到5毫升圆底管中。 </li></ol></li><li> 从组织培养物中收获细胞 <ol><li>洗涤板或贴壁细胞在室温下用钙和镁的磷酸盐缓冲盐水（PBS）的烧瓶中。 注意:用于获取细胞中记载的技术是专门适用于贴壁的人胚胎干细胞（hESC细胞）培养细胞。然而，所描述的协议的其余部分是广泛适用于其它培养的细胞系，非粘附系和原代细胞。 </li><li>添加温热（37℃）的胰蛋白酶或其他酶消化试剂用于实现从所述贴壁细胞的单一细胞悬浮液进行了优化。按照制造商的协议。 注:胰蛋白酶等酶分解试剂具有影响细胞标志物的潜力。 </li><li>将培养板在37°下2-5分钟，以分离细胞。对于高汇合文化，轻轻敲击板以帮助分离细胞。 </li><li>传送完全分离的细胞从板至5mL圆底管中，用1毫升移液管以解离细胞轻轻吸管。 注意:为了处理大量的样品可以替代在96孔圆底板中进行所有步骤。对于在96孔格式处理的样品的所有清洗液会在250μL的体积来进行。在其他步骤中描述的体积可被调整，从而总体积不超过300μL。 </li><li>淬火用等体积的洗涤缓冲液（0.5％BSA和0.02％叠氮化钠于PBS中）的酶促消化试剂。 注:叠氮化钠是一种危险化学品。适当的安全防范措施必须为它的制备和处理进行观察。 </li><li>离心细胞于在37℃下300×g离心5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>重悬细胞在1mL无血清培养基。 </li><li>通过转移的细胞悬浮液10微升到1.5mL微量离心管中计数细胞。稀释等分试样，以台盼蓝和转移到血球或者自动细胞计数系统中的已知比率。 </li></ol></li><li> S期人口标签 注:如果S-期标签是不进行继续干1.4。 <ol><li>在无血清的DMEM F12或其他适当的介质制备1毫升10μM5-碘-2&#39;-脱氧尿苷的（IDU）用于待分析的每个2×10 6个细胞。 </li><li>在无血清培养基中添加500μL的10个μMIDU对于每个2×10 6个细胞。 注意:虽然IDU标记可以在含血清培养基中进行，无蛋白质将与顺铂的反应，防止死细胞的适当的标签。如果IDU期间使用标记含血清培养基的额外洗涤步骤是无血清培养基是必要的顺铂活/死STA之前去除血清蛋白都进不去。 </li><li>轻轻重悬用1mL移液管粒料。 </li><li>孵育在37℃下10分钟，轻微摇动。 </li></ol></li><li> 活/死标签 <ol><li>对于每个样品制备50μM的顺铂的500微升无血清的DMEM-F12或细胞类型适当的介质。 注意:如果由IDU S相人口的标记不被执行，以4×10 6细胞/ mL的浓度再悬浮的样品。重悬细胞加顺铂能够均匀活/死的标签解决方案的快速混合前。 </li><li>添加每2×10 6个细胞50μM顺铂的500微升样品中。 </li><li>在室温（RT）1分钟孵育上持续混合的轨道摇床。 </li><li>猝灭顺铂与等体积的洗涤缓冲液中。 </li><li>离心细胞，在300×g下在室温下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>洗两次样本用500μL洗涤缓冲液以除去过量的顺铂。 </li><li>洗涤样品用500微升PBS洗涤两次以除去过量的蛋白质。 </li></ol></li><li> 样品固定 <ol><li>在500μL的PBS中重悬样品。 </li><li>添加4％PFA的等体积的在PBS中的2％的终浓度吸管混合。 注:从准备粉末4％PFA新鲜，调节pH至6.9并通过一个0.44微米的过滤器。在PBS中的4％PFA可以被存储在4℃下长达两周。避免在安瓿供给预制福尔马林，除非专门测试，因为它常常包含可能与所测量的信道质量干扰金属污染物。 PFA的较低浓度可足以并有助于减少固定的对抗体结合的影响，但应根据经验测试。 </li><li>在室温下孵育15分钟，在持续混合的轨道摇床。 </li><li>离心细胞，在300×g下在4℃下5分钟。倒出或仔细吸出上清液无线thout搅动沉淀。 </li><li>洗液样品用PBS以除去PFA溶液。 注:在此步骤的样品可以简要地在4℃下储存。在4℃下长期储存，可能导致样品的降解和减少的染色。 </li></ol></li></ol> 2.条形码注意:尽管同时呈现方法用于利用单同位素顺铂条形码和条形码钯，或者可以独立地使用，或如果不通过实验所需完全避免。 <ol><li> 同位素顺铂条形码 注:由于顺铂条形码与顺铂活/死染色依靠重叠信道应小心，以确保在活细胞这样的背景下顺铂活/死染色最小化，因为这可能与顺铂条形码的准确性干扰。 <ol><li>稀释1毫米同位素顺铂原液在PBS 200微米。 </li><li>前面ACH独特顺铂条形码制备1.5mL管用500μl的PBS对每个2×10 6细胞接收条形码。 </li><li>为了制备每个独特的条形码的每个适用的单同位素顺铂添加至200nM的终浓度。 </li><li>将细胞重悬在每2×10 6个细胞500μl的PBS中。 </li><li>添加相应的条形码溶液到每个样品的等体积。 </li><li>在室温下孵育样品5分钟上持续混合的轨道摇床。 </li><li>猝灭顺铂与等体积的洗涤缓冲液中。 </li><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>洗液样品用500μl洗涤缓冲液两次，以除去过量的顺铂。 </li></ol></li><li> 钯条形码 注:钯条形码试剂可以是准备好的5或商购。 <ol><li>瞬态部分通透19 注:钯螯合条形码需要局部透的试剂穿过细胞膜和鲁棒标记细胞。 <ol><li>洗涤样品用500微升PBS中。 </li><li>洗涤样品用500μL的PBS加0.02％皂苷。 </li><li>重悬沉淀在剩余体积。 </li></ol></li><li>应用钯条形码。 <ol><li>稀钯条形码试剂的100倍的库存在1mL冰冷的PBS加0.02％皂苷。 </li><li>迅速加稀条形码试剂再悬浮样品。 </li><li>在持续混合在室温下孵育在轨道振荡器上15分钟。 </li><li>洗液样品用500μL两次洗涤缓冲液。 </li></ol></li></ol></li></ol> 3.细胞表面染色注:细胞表面染色可以在固定之前活细胞来进行。这对于失去亲和力THEI抗体是必要的固定之后的R表位。一些细胞样品可受益于附加的阻挡，例如Fc阻断白细胞之前，抗体标记以减少背景。最佳阻挡应该针对特定样品类型根据经验来确定。 <ol><li> 制备的细胞表面抗体染色面板。 <ol><li>结合0.5μL，或根据经验确定的量，每个样品各0.5mg / mL的细胞表面抗体，到1.5mL管中。添加洗涤液如果有必要把音量高达每样10μL。通过移液混合。 注:确定由滴定实验凭经验实验前各抗体工作稀释度。 </li><li>分裂的细胞表面抗体池等分为一个1.5mL管中对每个样品被染色。 </li><li>制备500μl的洗涤缓冲液在1.5mL管中的每个样本。 </li></ol></li><li> 应用细胞表面染色样品在标准体积的所有样本。 注:要ENS在多个样品URE一致抗体标记是至关重要小心地控制样品体积和抗体浓度。下面的步骤旨在通过确保抗体已经被添加后的最终样品体积是均匀的，以实现这种一致性。 <ol><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>用200微升移液管组50μL，移除样本颗粒和转印剩余溶液到等分的细胞表面抗体池的管。 </li><li>吸取了在等分试样管中的所有液体而不将空气吸入到移液管尖端。 </li><li>而保持移液管柱塞，以避免吸入空气移动尖端插入的500μL准备好的管洗涤缓冲液和释放柱塞。 </li><li>移液管尖端的内容添加回样品重悬在温和移液液体的样品。 </li><li>孵育1样品上持续混合的轨道摇床在RT小时。 </li><li>洗液样品用500μl洗涤缓冲液两次，以确保所有游离抗体被洗掉。 </li><li>洗涤样品用500微升PBS中。 </li><li>重悬在500μlPBS中的细胞沉淀。 </li><li>添加在PBS中的4％PFA的等体积;吸管混合。 </li><li>孵育在轨道振荡器上10分钟。 </li></ol></li></ol> 4.细胞透化和染色细胞内<ol><li> 细胞透 <ol><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>通过温和涡旋直到沉淀解离重悬残留体积的细胞。 注意:如果不分离之前加入的MeOH将导致细胞粘附在一起，并且产生薄膜，这将导致部分样品损失的细胞。 </li><li>立即加入1每2×10 6 100％MeOH中&lt;/ SUP&gt;细胞，轻轻移液管混合。 注意:其他通透方法可以代替MeOH和可能是必要的，以实现最佳透对于某些类型的细胞。对于一些细胞源的0.1％的Triton X-100，在透化的PBS溶液0.2％Tween-20的或0.1％皂苷可保持细胞完整性更好，同时仍然提供一致的透化。 </li><li>孵育样品至少1小时或最多24个小时，在4℃下透化。 注:在此步骤在MeOH样品可在-80℃保存长达1个月。 </li><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>加入1毫升的洗涤缓冲液对每个毫升MeOH中用于透化样品。轻轻重悬细胞沉淀。 </li><li>洗样品两次用500μL洗涤缓冲液。 </li></ol></li><li> 准备细胞内抗体染色面板。 <ol><li>结合0.5μL，或根据经验确定的量，每个样品各0.5mg / mL的细胞内抗体的，到1.5mL管中。添加洗涤液如果有必要把音量高达每样10μL。通过移液混合。 </li><li>分裂细胞内的抗体池等分为一个1.5mL管中的每个样品。 </li><li>制备洗涤缓冲液500微升在1.5mL管中的每个样本。 </li></ol></li><li> 在标准体积的所有样本应用细胞内染色样品。 注:为了确保在多个样品一致抗体标记是至关重要小心地控制样品体积和抗体浓度。 <ol><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>用200μl移液管设置为50μL去除剩余的从样品颗粒和转印解决等分细胞表面抗体池的管。 </li><li>吸管了所有在等分试样管液体而不将空气吸入到移液管尖端。 </li><li>而保持移液管柱塞，以避免吸入空气移动尖端插入的500μL准备好的管洗涤缓冲液和释放柱塞，编制洗涤缓冲液为50μL的总样品体积。 </li><li>移液管尖端的内容添加回样品重悬在温和移液液体的样品。 </li><li>孵育在持续混合的轨道摇床在RT 1个小时的样品。 </li><li>洗液样品用500μL两次洗涤缓冲液。 </li><li>洗涤样品用500微升PBS中。 </li></ol></li></ol> 5.铱标签<ol><li> 修复样本 <ol><li>重悬的样品在PBS中的500μL。 </li><li>添加4％PFA的500微升PBS中。 </li><li>在室温下孵育至少15分钟，在持续混合的轨道摇床。 </li></ol></li><li> 应用DNA的铱标记 <ol><li&gt;稀释在PBS中的4％PFA的125μM500X铱嵌入试剂62.5 nM的浓度。 </li><li>加入500微升62.5纳米铱PFA解决方案样品。 注:对于使用透化的细胞的实验中，稀释500X Ir191 / 193股票为1的最终稀释:2000，以避免overstaining。 </li><li>在室温下孵育15分钟，在持续混合的轨道摇床。 注:如果在执行单同位素顺铂条形码不孵育与铱的样品的时间超过15分钟，因为强Ir193信号可以向Pt194质量和信道质量条形码产生负面影响。 </li><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li><li>洗涤样品在过滤的去离子水500μL0.1％BSA的两倍。 注意:一旦准备好，我们建议尽可能在24个小时内运行样品。制备的样品可以在4℃保存短期内，但应注意避免降级。如果可能最终洗涤应在过滤的去离子水，无BSA进行，因为这将降低在其上会降低仪器的清洁机器的喷雾室和采样器锥体的沉积。对于胚胎干细胞，有必要同时包括BSA，以避免粘到管表面的样品损失来执行这些洗涤。 </li></ol></li></ol> 6.准备样品采集<ol><li> 细胞计数 <ol><li>将细胞重悬在500μL的0.1％BSA在过滤的去离子水，并通过35微米的细胞滤网过滤帽到一个新的试管中。 注:降低了BSA水平，为最终降低洗涤残留物留在大众雾化仪。非贴壁细胞可洗涤并重新悬浮在过滤的去离子水。 </li><li>通过转移的细胞悬浮液10μL至微量离心管中计数细胞。稀释等分试样以已知比率台盼蓝（与细胞可视化助剂）并转移至血细胞计数器或自动细胞计数系统。 </li><li>离心细胞，在300×g下在4℃下5分钟。倾析或小心吸出上清液，而不会干扰沉淀。 </li></ol></li><li> 准备和装入样品 <ol><li>为了准备校准珠从冰箱中取出，并剧烈振荡重悬。 </li><li>如果运行使用质量自动进样器流式细胞术样品中，添加50微升的校准珠的样品板的每个孔中再加入细胞的期望数量进行分析。如果通过手动注射在运行样品添加的校准珠50μL样品，稀释在过滤的去离子水样品，拌匀和负载样品放入流式细胞仪质谱样品注射端口。合适的样品稀释可根据细胞类型进行分析而变化，但10 6稀释一般是适当1。 </li></ol></li></ol>

<ol>
	<li>Leipold, M. D., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+cytometry:+protocol+for+daily+tuning+and+running+cell+samples+on+a+CyTOF+mass+cytometer.">Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer.</a> J Vis Exp. , e4398 (2012).</li><li>Leipold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+step+on+the+path+to+mass+cytometry+standardization.">Another step on the path to mass cytometry standardization.</a> Cytometry A. 87, 380-382 (2015).</li><li>Newell, E. W., 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=Combinatorial+tetramer+staining+and+mass+cytometry+analysis+facilitate+T-cell+epitope+mapping+and+characterization.">Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization.</a> Nat Biotechnol. 31, 623-629 (2013).</li><li>Leong, M. L., Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Peptide-MHC+Tetramer+Staining+with+Mass+Cytometry.">Multiplexed Peptide-MHC Tetramer Staining with Mass Cytometry.</a> Methods Mol Biol. 1346, 115-131 (2015).</li><li>Zunder, E. R., 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=Palladium-based+mass+tag+cell+barcoding+with+a+doublet-filtering+scheme+and+single-cell+deconvolution+algorithm.">Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.</a> Nat Protoc. 10, 316-333 (2015).</li><li>Lai, L., Ong, R., Li, J., Albani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD45-based+barcoding+approach+to+multiplex+mass-cytometry+(CyTOF).">A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).</a> Cytometry A. 87, 369-374 (2015).</li><li>Mei, H. E., Leipold, M. D., Schulz, A. R., Chester, C., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoding+of+live+human+peripheral+blood+mononuclear+cells+for+multiplexed+mass+cytometry.">Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.</a> J Immunol. 194, 2022-2031 (2015).</li><li>Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platinum-based+covalent+viability+reagent+for+single-cell+mass+cytometry.">A platinum-based covalent viability reagent for single-cell mass cytometry.</a> Cytometry A. 81, 467-475 (2012).</li><li>Krutzik, P. O., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cell+barcoding+in+flow+cytometry+allows+high-throughput+drug+screening+and+signaling+profiling.">Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.</a> Nat Methods. 3, 361-368 (2006).</li><li>Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+adapted+to+measurements+of+the+cell+cycle.">Single-cell mass cytometry adapted to measurements of the cell cycle.</a> Cytometry A. 81, 552-566 (2012).</li><li>Edgar, L. J., 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=Identification+of+hypoxic+cells+using+an+organotellurium+tag+compatible+with+mass+cytometry.">Identification of hypoxic cells using an organotellurium tag compatible with mass cytometry.</a> Angew Chem Int Ed Engl. 53, 11473-11477 (2014).</li><li>Bendall, S. C., 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=Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum.">Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.</a> Science. 332, 687-696 (2011).</li><li>Horowitz, 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=Genetic+and+environmental+determinants+of+human+NK+cell+diversity+revealed+by+mass+cytometry.">Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry.</a> Sci Transl Med. 5, 208ra145 (2013).</li><li>Zunder, E. R., Lujan, E., Goltsev, Y., Wernig, M., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+molecular+roadmap+to+iPSC+reprogramming+through+progression+analysis+of+single-cell+mass+cytometry.">A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry.</a> Cell Stem Cell. 16, 323-337 (2015).</li><li>Han, L., 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=Single-cell+mass+cytometry+reveals+intracellular+survival/proliferative+signaling+in+FLT3-ITD-mutated+AML+stem/progenitor+cells.">Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.</a> Cytometry A. 87, 346-356 (2015).</li><li>Bendall, S. C., 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=Single-cell+trajectory+detection+uncovers+progression+and+regulatory+coordination+in+human+B+cell+development.">Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development.</a> Cell. 157, 714-725 (2014).</li><li>Qiu, P., 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=Extracting+a+cellular+hierarchy+from+high-dimensional+cytometry+data+with+SPADE.">Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE.</a> Nat Biotechnol. 29, 886-891 (2011).</li><li>Amir el, A. D., 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=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nat Biotechnol. 31, 545-552 (2013).</li><li>Behbehani, G. K., 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=Transient+partial+permeabilization+with+saponin+enables+cellular+barcoding+prior+to+surface+marker+staining.">Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.</a> Cytometry A. 85, 1011-1019 (2014).</li><li>Finck, R., 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=Normalization+of+mass+cytometry+data+with+bead+standards.">Normalization of mass cytometry data with bead standards.</a> Cytometry A. 83, 483-494 (2013).</li><li>Chester, C., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algorithmic+Tools+for+Mining+High-Dimensional+Cytometry+Data.">Algorithmic Tools for Mining High-Dimensional Cytometry Data.</a> J Immunol. 195, 773-779 (2015).</li><li>Diggins, K. E., Ferrell, P. B., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+discovery+and+characterization+of+cell+subsets+in+high+dimensional+mass+cytometry+data.">Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data.</a> Methods. 82, 55-63 (2015).</li><li>Leelatian, N., Diggins, K. E., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Phenotypes+and+Signaling+Networks+of+Single+Human+Cells+by+Mass+Cytometry.">Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.</a> Methods Mol Biol. 1346, 99-113 (2015).</li><li>van der Maaten, L. J. P., Hinton, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizaing+Data+using+t-SNE.">Visualizaing Data using t-SNE.</a> J. Mach. Learn. Res. 9, 2431-2456 (2008).</li><li>Kling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry:+Measure+for+measure.">Cytometry: Measure for measure.</a> Nature. 518, 439-443 (2015).</li><li>Gregori, G., 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=Hyperspectral+cytometry.">Hyperspectral cytometry.</a> Curr Top Microbiol Immunol. 377, 191-210 (2014).</li><li>Chattopadhyay, P. K., 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=Brilliant+violet+fluorophores:+a+new+class+of+ultrabright+fluorescent+compounds+for+immunofluorescence+experiments.">Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments.</a> Cytometry A. 81, 456-466 (2012).</li><li>Rieger, A. M., Havixbeck, J. J., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-FISH:+Analysis+of+cellular+RNA+expression+patterns+using+flow+cytometry.">X-FISH: Analysis of cellular RNA expression patterns using flow cytometry.</a> J Immunol Methods. 423, 111-119 (2015).</li><li>Frei, A. P., 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=Highly+multiplexed+simultaneous+detection+of+RNAs+and+proteins+in+single+cells.">Highly multiplexed simultaneous detection of RNAs and proteins in single cells.</a> Nat Methods. 13, 269-275 (2016).</li></ol>

The authors have nothing to disclose.

这里介绍的协议已被成功地用于各种培养的细胞系（H1和H9胚胎干细胞，mESCs，MCF7，HEK 293，KBM5，HMEC，MCF10A）和伯组织样品（小鼠骨髓，小鼠胚胎肝，小鼠成年的处理肝，小鼠肿瘤）。不管源，可以解离成单细胞，同时保留所述蜂窝状态应该是适合于通过质量分析仪，但可能需要一些协议优化任何组织。需要注意的是一些表位可以通过使用特定的解离，固定和渗透的治疗受到影响，这些方法可能需要经过样品特定的优化是很重要的。如果低信号被用于抗体观察到的有可能通过对活细胞进行的细胞表面标记，改变透方法，或降低定影时间来提高。 如果可能的实验设计中，包括在第条形码È样品处理工作流在细胞和抗体浓度的水平，确保样本之间的一致性。没有条码，即使当非常小心，以控制样品处理，则很可能在抗体浓度和细胞浓度的微小变化将样品之间被引入。此外，由于细胞的阳性特异性表位的百分比很可能样品之间变化时，有效的抗体对表位的比率可以不一定样品之间不同，即使总抗体浓度保持一致，从而可能导致在抗体染色不一致。这些变型中，观察单个信道时同时一般轻微的，可以执行高参数实验和如果不注意数据分析过程中采取可被误解为生物学上有意义时变得大大加剧。 从一个大容量实验仪得到的数据的分析可以采取许多形式，并且一个数computa的周志武方法已被用于这种应用特别适合。虽然质量提供的有用的算法的详尽的调查术数据的分析已超出了本手稿的范围内，已被成功地使用的方法被高亮显示感兴趣的读者涉及多个资源覆盖本主题21，22，23。的大规模术数据的分析中最广泛采用，目前可用的方法有两种密度跨越树进展标准化活动（SPADE）和viSNE。 SPADE从具有类似标志物表达的细胞群形成簇和表示每个集群作为一个节点。这些节点被安排在其中类似节点通过线（ 图3G），其连接一个SPADE树。 viSNE利用叔分布式随机邻居嵌入算法24来生成的二维表示高维数据，同时保持总体数据结构。无论铁锹viSNE都写在MATLAB，源代码是开放的，并且可以通过MATLAB用户运行。此外，黑桃的独立版本。 虽然大众目前流式细胞允许的参数数量最多的集合，基于荧光标记流式细胞仪可对一些应用程序更好的方法。荧光流式细胞仪可以分析更多的细胞每秒，至多5×10 4相比为500-1000质量流式细胞术，并已更多可用验证抗体25。荧光流动的附加进步仪，如光谱仪26和新的荧光团27构成荧光流式细胞一个可行的选择，但所有非常高参数的实验。另外的技术，例如RNA 28的测量以前只可能通过基于荧光流式细胞术最近已适应于大量术允许蛋白质和RNA 29的同时检测。可用同位素和试剂的未来扩展到更完全地利用可测量的同位素的质量窗口和扩大的可测量的细胞特性的剧目将扩大质量术剖析可能的深度。 大量的可以同时由质量术分析参数大大扩展了可待研究的实验问题。我们希望这篇文章和相应的视频协议将使感兴趣的研究人员更有效地利用流式细胞仪质量，以促进他们的研究。

流式细胞术使多个抗体靶的同时测量在跨越大量人口细胞的单细胞水平。在传统的基于荧光的流式细胞仪中，可以量化参数的数量，由多个荧光团的发射光谱，这需要日益复杂的补偿计算作为参数的数量增加时之间的光谱重叠限制。这些限制由质量流式细胞术，其中检测和通过飞行时间（TOF）质谱法的时间，大大扩大的同时收集的参数的数量和产生高维蛋白丰度分布为每个单独的小区量化重金属结合的抗体处理。 质量血细胞计数仪的工作的基本的理解是有利于用户，并且可以是用于故障诊断至关重要。用于调谐的更透彻的描述和运行质量术机，请参阅相关的手稿1。简言之，将细胞样品标记有靶向细胞表面标记物，细胞质蛋白，核蛋白，染色质结合的蛋白质或感兴趣（ 图1i）中其它表位金属缀合的抗体的面板。将标记的细胞被装载到机器上，或者在一个通过手动注射时间或使用自动进样器，其从96孔板的样品。所加载的细胞通过喷雾器，其产生液滴包封细胞（ 图1II）的喷雾喷射。这种喷雾被定位成使得所述细胞通过在氩等离子炬电离。这种电离产生由每个小区（ 图1iii）的所有构成原子的粒子云。作为这种粒子云行进到所述检测器，低原子质量原子从高质量离子通过四极质量过滤器分离。剩余的高质量离子继续到检测器，其中所述abund各同位素的ANCE进行定量（ 图1iv）。由检测器收集的原始数据，由质量血细胞计数仪软件分析，以确定细胞事件。对于每一个识别的小区时，在每个信道检测的信号进行量化，并保存到输出.fcs文件。用于检测重金属缀合抗体的质量信道表现出最小的光谱重叠，其中从0-4％的范围内具有低于1％，最贡献。因为信道之间的这种低的串扰，所以一般不需要分析2之前与补偿矩阵来转换数据。然而，应注意的抗体的实验面板的设计期间进行，以确保具有高丰度表位的抗体没有分配给有助于信号分配给一个低丰度抗体的信道的质量的信道，因为这可能创建在信道接收所述信号贡献人为的高人口。 <p cl屁股="jove_content"&gt;大规模仪分析样品的确切制剂是高度依赖于类型的样品被收集和实验假设正在测试中。我们提供的两个协议实例用于收获的不同类型的细胞（人胚胎干细胞）和原代细胞（小鼠乳腺肿瘤上皮细胞分离物）。当开始大规模研究流式细胞术，最好是先进行小试实验，以确保所有的协议和抗体被用来在研究者手中运作良好。这是特别必要时要使用的自定义缀合抗体，该抗体缀合过程中的部分还原反应有可能破坏抗体亲和力的潜力;因此，每个自定义抗体需要凭经验验证，以确保数据的准确性。其他考虑因素包括:确定在测定中使用固定之后将认识到它们的表位的细胞表面的抗体，或者如果他们NE要被施加到编活细胞。如已知与肽-MHC染色3,4发生一些固定可以阻止正常染色与细胞表面的抗体。活细胞上进行细胞表面抗体染色可以针对一些抗体是必要的，但是这排除，因为大多数条形码方法固定后5虽然基于抗体的条形码6中 ，7是一个例外执行之前抗体标记条形编码的选项。 当确定细胞的数目，以用于分析来制备，重要的是要知道，只有加载到机器中的细胞的一小部分将被检测到，并产生数据。这种损失主要是由于低效当喷雾器喷洒在焊炬的样品。确切的损失百分比取决于质量流式细胞机设置和细胞类型，但范围可以从50-85％，并且要设计实验时考虑。该协议具有用于每个样品2×10 6个细胞被优化，但可适合于处理更小或更大的样本大小。该协议可以以最小的修改来用于样品尺寸从1×10 6至4×10 6，但它是非常重要的样本大小被保持一个实验内是一致的。细胞数目的变化可影响条形码和抗体染色的强度和对面直接比较样品应保持大致一致的。 一些程序，如死细胞标记和DNA标记，应在绝大多数的进行，如果不是全部的实验设计。可使用Rh103实现死细胞的实验中仅分析表面标记的标签，但Rh103应避免其中，因为它导致染色损失需要透实验。对于涉及通透试验，最好是利用顺铂<s向上类="外部参照"&gt; 8，并且在可能时识别切割的PARP或裂解的caspase的抗体检测凋亡和死细胞。 条形码的样品可以降低抗体的消耗，减少捕获时间和消除样品到样品的可变性9。条形码的方法，涉及将唯一的特定示例代码的所有小区，这是用来给每个小区分配到其起源的样品。条形编码样品后可先于抗体染色，以确保所有样品被相等地染色的过程中合并。为了最大限度地减少在实验误差，它是明智的条形码和池将被直接相互比较的任何样品。目前存在用于条形编码样品质量仪分析5，6，7，其中两个是这里提出（见下文）的几种方法。 目前可用reagenTS有可能量化的38个抗体目标的丰度，确定在S期10个细胞，区分活细胞和死细胞8和多个样本的复用实验测量缺氧11的细胞水平。此收集高参数单细胞数据大细胞群体的能力能够改进高度异质的细胞群的分析，并且已经产生了一些新的生物洞察12，13，14，15，16的。蒸馏所产生的复杂的数据可解释信息需要运用的计算算法包括密度归一化活动的生成树进展（SPADE）17和18 viSNE。 本文介绍了访问的概述如何设计和进行质量流式细胞实验，并引入质量术数据的基本分析。

<table><tbody><tr><td>mTeSR1 medium kit</td><td>Stem Cell technologies</td><td>05850</td><td>Warm at room temperature before use</td></tr><tr><td>DMEM F-12</td><td>ThermoFisher</td><td>11330-032</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Accutase</td><td>Stem Cell technologies</td><td>07920</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Bovine serum albumin</td><td>Equitech</td><td>BAH62</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Cell ID Pt194</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt195</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt196</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cisplatin</td><td>Enzo Life Sciences</td><td>ALX-400-040-M250</td><td>Soluble to 25&amp;nbsp;mg/mL in DMSO</td></tr><tr><td>Cell-ID 20-plex Pd Barcoding Kit</td><td>Fluidigm</td><td>201060</td><td></td></tr><tr><td>5-Iodo-2&#039;-deoxyuridine</td><td>Sigma-Aldrich</td><td>I7125</td><td>Soluble to 74&amp;nbsp;mg/mL in 0.2&amp;nbsp;N NaOH</td></tr><tr><td>Rhodium 103 intercelating agent</td><td>Fluidigm</td><td>201103A</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>BP1105-4</td><td>Chill at -20&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>5 mL round bottom tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5 mL 35 &amp;#956;m filter cap tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>EQ four element calibration beads</td><td>Fluidigm</td><td>201078</td><td></td></tr><tr><td>Hyaluronidase Type I-S</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Disposable Scalpel #10</td><td>Sigma-Aldrich</td><td>Z69239</td><td></td></tr></tbody></table>

sample preparation for mass cytometry analysis

质谱术利用具有重金属的标签，这种方式大大增加了参数和深入的分析机会的数量远远超出什么是可能的流式细胞术常规的基于荧光的偶联流抗体。正如任何新技术，有利于保证可靠的新一代高品质数据的关键步骤。这里介绍的是一个优化的协议了采用多种技术用于细胞样品的大规模细胞仪分析的处理。这里所描述的方法将帮助用户避免常见的陷阱，并通过减少可变性，从而导致不准确的数据实现一致的结果。为了通知实验设计，在后面的协议和它们在被调查系统的生物学发现新的发现功效任选的或可替代的步骤的理由被覆盖。最后，有代表性的数据以说明从技术presente预期效果这里天。

这里介绍的协议可以被广泛地应用到各种各样的培养和主细胞样品，只有轻微的修改。根据实验的要求，可以以模块化的方式时的某些元件，如条形码或细胞表面染色，是没有必要进行。使用其全部这个协议使得多路复用块的标记的流式细胞术对死细胞排除，S相人口识别和染色的高达38个抗体靶向所述量化样本的制备。 通过在这个协议中（ 图2）中概述的方法产生的预期的结果在图3中被描绘。当收集数据，基于该校准珠强度信号强度的归一化可以质量流式细胞术软件中执行。遵循规范化，初始数据的凝固酶原可以手动使用能够分析流式细胞术数据的任何软件来执行ssing以去除单个细胞校准珠，栅极和去除死细胞。的校准珠除去可以手动通过门控的Ir191 + Ce140-人口（ 图3A）或用基于MATLAB算法20来执行。单细胞事件（黑色轮廓， 图3B）可以同时除去碎片和细胞多重峰，通过门控Ir193和事件长度（ 图3B）的双轴情节被选通。死细胞的顺铂标记可用于定量细胞死亡的量;示出的是具有低（ 图3D）和死细胞的更高（ 图3E）量的样品的例子。死细胞可以从数据通过门控关Pt198 +群体（ 图3E）被移除。样品的条形码，不管是用单一同位素顺铂或钯试剂，导致Sa的不同分离的条形码参数（ 图3C），其允许细胞内mples要被分配给他们的原始样品的身份。附加的可选方法，如IDU标签，允许特定人群， 例如检测，S相的细胞（ 图3F）。以下初始正常化，debarcoding和选通，高维数据可以使用各种算法，包括SPADE 17和其他几个设计用于大量术数据的分析和可视化18进行分析。算法，如SPADE采取高维数据，其可以是难以解释，因为它不能直接在其高维状态可视化，并且生成数据的一个低维表示，这是更易于视觉解释（ 图3G）。 <img alt="图1" src="/files/ftp_upload/54394/54394fig1.jpg"/&gt; 图1: 流式细胞仪的质量测量的基本原理。细胞样品的大规模细胞仪分析制备涉及染色的细胞用标记有来自镧系元素的金属多为同位素的抗体。在这个方案中针对特定表位的凸起每个抗体用同位素具有一个独特的原子质量，如钐154抗体针对那些表面，细胞质，可潜在地用于局部核染色质或表位。标记的细胞被注入并通过质量仪喷雾器为其中它们被氩等离子体炬电离单细胞的液滴喷射。而较大的原子质量的离子的丰度是由检测器测得的所得粒子云被剥离通过四极质量过滤低质量的原子。每个唯一的质量离子的测定量对应于与其相关联的抗体的目标表位的细胞丰度。虽然理论上升超过100的参数可以IMIT利用该方法来定量，目前市售的试剂允许每个小区40的参数的检测。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54394/54394fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54394/54394fig2.jpg" /> 图2: 实验步骤和任选的步骤的工作流程图。中涉及的协议制备细胞质量流式细胞术的主要步骤描述。可选步骤用橙色框标记。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54394/54394fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54394/54394fig3.jpg" /> 图3: 从 米 屁股仪 分析 产生的数据 的实施例 。 （A）示出的细胞（高Ir191，低Ce140），小珠的分离（低Ir191，高Ce140）和胎圈芯的双峰（高Ir191，高Ce140）双轴曲线图。 （B）双轴积表示VS事件长度Ir193的预期外观。单个细胞的门控其除去细胞碎片和细胞多重峰被示出。 （C）双轴积表示选自顺铂条形码两个质量信道的示例图。 （D - E）选自顺铂活/死染色实施例的数据与低百分比（D）和死细胞的高百分比（E）的样品。 （F）与IDU标记来区分在S期细胞的hESC H9细胞的双轴情节和抗细胞周期蛋白B1的抗体，以进一步区分细胞周期。 （G </st从35抗体质量流式细胞仪的hESC的实验和从蛋白质标记构建和由丰度所测量的参数中的一个的色的代表SPADE树数据的荣&gt;）N×N个图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54394/54394fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Sample Preparation for Mass Cytometry Analysis

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

sample-preparation-for-mass-cytometry-analysis

Universidad San Sebastian

Research

JoVE Journal

Biology

16.0K 次观看.  The University of Texas MD Anderson Cancer Center. This article describes the collection and processing of samples for mass cytometry analysis.

视频: Sample Preparation for Mass Cytometry Analysis

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

科研

生物工程

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

发育生物学

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

医学

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

免疫与感染

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

小鼠细胞类型的低起始量代谢研究

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

作者聚焦:推进稀有造血干细胞的代谢组学分析 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

罕见原代细胞的靶向代谢组学

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

样品制备质谱术分析

摘要

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此视频中的章节

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

收集，分离，和人类宫颈样品的流式细胞仪分析

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

罕见原代细胞的靶向代谢组学

罕见原代细胞的靶向代谢组学

罕见原代细胞的靶向代谢组学

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因的体外分化

样品制备质谱术分析

摘要

探索更多视频

此视频中的章节

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

收集，分离，和人类宫颈样品的流式细胞仪分析

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

罕见原代细胞的靶向代谢组学

罕见原代细胞的靶向代谢组学

罕见原代细胞的靶向代谢组学

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因 的体外 分化

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因的体外分化