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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#176;C before use</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell technologies</td>
			<td>07920</td>
			<td>Warm at 37&#176;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 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt195</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt196</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-400-040-M250</td>
			<td>Soluble to 25mg/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&#39;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>I7125</td>
			<td>Soluble to 74mg/ml in 0.2N 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&#176;C before use</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml round bottom tubes</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml 35um 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?...

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

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

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.

对于外小泡的使用流式细胞仪分析技术

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

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...