We thank Lucile Wrenshall for the use of the plate reader, Nancy Bigley for useful discussion, and Tom Brown for the gift of ICR mice. This work was funded by grant 1R01AI114804 from the National Institute of Allergy and Infectious Diseases and by WSU Faculty Development funds (to J.A.K.). J.N.G. was supported in part by the National Institute of General Medical Sciences grant R25GM090122.

所有实验均按照由莱特州立大学实验动物护理和使用委员会和机构审查委员会批准的协议进行的。 注意:人PBMC通过Ficoll密度梯度离心法5分离。 1.脾虚收获<ol><li> 房子成年雌性ICR小鼠与食品，水和被褥特定于物种要求标准实验室条件下。 注:动物有2.9个月，平均年龄和30.4 g的安乐死的时间平均重量。 </li><li>在隔离（DOI）之日起，安乐死的动物没有单独存在的其他同种动物。尽一切努力，以确保最小的压力鼠标。用安乐死的 CO 2继发颈椎错位，以确保死刑。 <ol><li>将动物，还是转移笼子里，入二氧化碳室和STA室温以5升/分钟的气流。该流速在每分钟推荐10-30％置换氧气。 </li><li>动物是无意识后，增加的 CO 2流速15升/分。检查动物呼吸停止，并在该腔室离开另外2分钟。 </li><li>适用于颈椎脱位伴扩张力，以确保死刑。 </li></ol></li><li> 收获使用死亡10分钟内无菌技术动物脾脏。 <ol><li>消毒两套剪刀和镊子的用于在高压釜使用烘箱之前的取出脾脏。通过应用70％乙醇消毒的剥离面。 </li><li>应用70％的乙醇对小鼠腹部。使用一组剪刀和镊子的，使得在从腹部的左侧的毛皮和皮肤的切口，然后拉开和剥离背部皮肤以显示腹膜。一旦接触，打开腹膜前应用4％洗必泰溶液。 </li><li>使用第二组脊髓损伤ssors和钳子，使沿中央腹部2至3厘米切口。打开腹膜并切除内脏附件和多余的脂肪取出脾脏。放置在Dulbecco磷酸盐缓冲盐水（DPBS）脾脏。 </li></ol></li></ol> 2.生脾细胞的制备注意:使用执行无菌技术层流生物安全柜下的所有步骤。 <ol><li> 浸泡收获脾脏在10ml去离子灭菌的 H 2的O 5分钟在10cm的培养皿以溶解表面红细胞。 注意:如果脾脏隔离过程中损坏该步骤可以省略。 </li><li>要释放脾，美眉2无菌载玻片之间的脾脏（磨砂面朝脾）在一个新的10厘米培养皿。用10毫升的RPMI-1640的补充有10％胎牛血清，2mM的L-谷氨酰胺，50IU / ml青霉素和50微克/ ml链霉素（下面称为一个混合氏完全RPMI）。 </li><li>过滤通过无菌40微米尼龙细胞过滤器的细胞进入一个新10厘米培养皿以除去结缔组织和碎片。 </li><li> 裂解通过加入20ml的RBC裂解缓冲液，在pH 7.4的155毫氯化铵 ，10毫碳酸氢钠 ，和0.1mM EDTA组成和〜300毫渗透摩尔浓度/升的其余红细胞。 注:渗透压由冰点或蒸汽压渗透压力计测定。 <ol><li>转移细胞从10厘米的板到50ml锥形管中并离心，在250×g离心10分钟（4℃）。 </li><li>除去上清液，添加20毫升RBC裂解缓冲液中，并通过移液重悬浮沉淀的细胞。 </li><li>在室温下孵育的裂解缓冲液10分钟，并轻轻摇动。 </li><li>离心机在250 xg离心（4℃）10分钟以沉淀细胞。倒出裂解缓冲液。 </li><li>重悬的细胞在10ml完全RPMI和离心机的试（250 XG，10分钟，4℃）。圆盘ARD上清液。 </li></ol></li><li>重新悬浮的脾细胞在2ml完全RPMI中温热至37℃，然后转移到尼龙羊毛纤维列。 </li></ol> 3. T细胞的分离纯化<ol><li> 用5毫升完全RPMI洗两次尼龙棉柱。在1小时，5％CO 2的细胞培养箱中在37℃。 注意:这是必须保持尼龙羊毛湿为实验的持续时间和使用的完全RPMI温热至37℃，所有的尼龙羊毛分离步骤。 </li><li> 孤立的脾细胞添加到列和孵育1小时，以使B细胞，成纤维细胞，和辅助细胞粘附到尼龙羊毛。 <ol><li>加2ml的RPMI含有脾细胞到柱并穿过直到达到尼龙羊毛的顶部。 </li><li> 2毫升完全RPMI中添加加热到37℃，在尼龙毛的顶部，并通过毛传递介质直到液面到达顶部表面。 </li><li>加入3毫升温完全RPMI中到塔以完全覆盖尼龙羊毛。 </li><li>孵育在37℃，5％CO 2的细胞培养箱中不受干扰1小时的装载柱。 </li></ol></li><li> 通过用5ml完全RPMI中洗涤柱两次洗脱的细胞。通过离心执行洗脱细胞的额外洗涤。 <ol><li>柱填充顶端2次完全RPMI，并允许在列中的溶液流过到50毫升的无菌锥形管中。 注:避免敲击或撞击之列，这可以撞出B细胞，辅助细胞和成纤维细胞附着在尼龙毛。 </li><li>收集在250 xg离心流过级分和自旋10分钟（4℃）使细胞沉淀。用10毫升完全RPMI洗一次。再次沉淀细胞（250 xg离心，10分钟，4℃）并在2毫升完全RPMI中重新悬浮。 </li><li>测量的C<html>使用ELL密度血球和在完全RPMI稀释到0.5×10 6个细胞/ ml。 </li><li>种子1或2萃取池的等分试样到24或6孔细胞培养板，分别的各孔中，并培养所述细胞在37℃，5％的CO 2。 注:1毫1,4-二硫苏糖醇（DTT）可添加到每个孔中在此阶段，提高T细胞的存活。 </li><li>可选:确认隔离协议通过流式细胞仪的效率。该协议通常产生〜80％T淋巴细胞23。 注:尼龙羊毛纯化细胞含有约50％ 的 CD4 +和23％的CD8 +细胞。以进一步纯化的CD4 +和CD8 +亚群，单免疫耗竭步骤可以使用八个抗体和磁珠24来执行。可替代地，更纯的CD4 +和CD8 + T细胞群可通过用特异性抗体阳性或阴性选择分离。 </li></ol></li></ol>_title"&gt; 4。T细胞的激活和实验药物暴露<ol><li>通过增加的PMA和离子霉素的钙盐或涂覆有抗CD3和抗CD28抗体23,25,26磁珠激活隔离的24小时之内的T淋巴细胞。在激活时添加试验药物（ 如，环孢素，FK506，雷帕霉素，TRAM-34，和FTY720）。 注意:在此，PMA浓度的2至250毫微克/毫升变化，并且离子霉素浓度保持在250纳米。如果可能的话，稀释应当从库存等分试样的每种药物，以使添加到细胞中的每个孔的体积为≥1微升，以确保可重复性制备。 1珠 - 细胞比:抗CD3 /抗CD28包被的磁珠在1中添加。增加每个细胞（ 即，胎圈至细胞比）小珠的数量将增加刺激25,26的强度。珠洗涤并再悬浮于完全RPMI它们的加入到细胞中之前。需要注意的是台盼蓝色污渍珠。 </li><li>培养在37℃，5％的CO 2与自动细胞计数器分析前将细胞12-72小时。 </li></ol> 5.自动细胞计数仪数据采集<ol><li> 运行示例之前，请确保细胞轻轻地用血清吸管，避免团块混合。这是因为它们的增加的密合性的活化的淋巴细胞特别重要。 <ol><li>对于与抗CD3 / CD28珠粒激活，吸液后培养，将样品转移到1.5ml或2毫升离心管中。通过保持管上的磁体1-2分钟分离珠。使用包含用于分析的细胞上清液。 注:分析1小时内两个静息和活化的细胞以考虑通过血清中存在的培养基中的静止细胞的任何预先活化。 PMA的12小时后/离子霉素刺激，细胞大小略有增加被检出（ 图2 </STRONG&gt;）。在72小时，比较无显著细胞大小增加，观察48小时（数据未显示）。 </li></ol></li><li> 传输1毫升细胞悬浮液到一个样品杯，并通过自动细胞计数器根据手册中的说明运行它。 注:对于每一个试验中，应该使用的最小的细胞培养物1毫升（最多2毫升）中。的自动细胞计数器使用的注射器与细胞悬浮液混合锥虫蓝和过该被成像和由软件进行分析的场传递细胞 - 台盼蓝混合物。软件检测台盼蓝染色的细胞，得出每个小区绕了一圈，并确定直径。 100图像从每个样品收集，以及细胞存活率和确定尺寸。这里使用的细胞计数器的检测阈值具有5微米的下限。 </li><li>从各传输数据运行，以供进一步分析的电子表格。 </li><li>校准设备上使用1定期为6μm和8μm的直径的聚苯乙烯珠毫升样品。 注意:由生产每批测定的实际珠尺寸应该使用，而不是标称尺寸。图6和8微米的珠是方便的，因为这些直径是接近静止的预期直径和活化的T细胞（参见图1）。 </li></ol> 6.数据和统计分析<ol><li> 使用合适的统计和图形软件分析数据。 注意:每次运行的电子表格中包含的细胞，每个直径数（范围:5微米到70微米）。一个17微米的直径以上的细胞从分析中删除排除粉尘颗粒和小气泡，这可通过在仪器被解读为活细胞。 </li><li>执行假设用韦尔奇的t检验数据集的方差不等测试;结果被认为如果p &lt;0.001统计学显著。使用的公式是， <im克ALT ="公式1"SRC ="/文件/ ftp_upload / 55212 / 55212eq1.jpg"/&gt; 哪里<img alt="公式（2）" src="/files/ftp_upload/55212/55212eq2.jpg" />和<img alt="公式3" src="/files/ftp_upload/55212/55212eq3.jpg" />是样本均值， <img alt="公式4" src="/files/ftp_upload/55212/55212eq4.jpg" />和<img alt="公式5" src="/files/ftp_upload/55212/55212eq5.jpg" />是样本方差，以及<img alt="公式6" src="/files/ftp_upload/55212/55212eq6.jpg" />和<img alt="公式7" src="/files/ftp_upload/55212/55212eq7.jpg" />是样本大小为每个数据集。的自由度的数目，使用两个样本大小为每个比较的小保守估计。条形图表示为平均值±SEM表示。 </li></ol> 7.脾T淋巴细胞增殖试验<ol><li> 使用MTS-或基于MTT比色读板器增殖测定测量T细胞增殖。 注:该法为基础在MTS四唑化合物（欧文的试剂）还原成可溶性甲产物，可能是由NADPH或NADH d在代谢活跃的细胞中的脱氢酶产生的。 </li><li> 计数纯化的T细胞与血球和在完全RPMI-1640以1×10 6个细胞/ ml的终密度重悬它们。 注:1毫摩尔DTT可在此阶段被加入到细胞中。 </li><li>激活用PMA的细胞并用试验药物离子霉素一起，如果需要的话，和轻轻混匀（类似于步骤4）。 </li><li>板100微升在96孔细胞培养物处理板的每孔的细胞，并在37℃下孵育，用5％CO 2的48小时。加入100微升的RPMI-1640培养基的无细胞进入一个孔，以获得背景吸光度的读数。 </li><li>加入20μl基于MTS的试剂至各孔，并在37℃，5％的CO 2孵育4小时。 </li><li> 用酶标仪采取在490nm吸光度测量。在一个bsorbance测量对应于每个代谢活性细胞的数量很好。 注:如有需要减去背景水平。背景吸光度值取决于培养基，血清，外部pH值，和MTS试剂暴露时间的光的类型。基于MTS-试剂对光敏感，并曝光了几个小时，可能会导致更高的背景吸光值。 </li></ol>

<ol>
	<li>Weiss, A., Samelson, L. E., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-lymphocyte+activation.">T-lymphocyte activation.</a> Fundamental Immunology. , 321-364 (2003).</li><li>Gergely, P., Ernberg, I., Klein, G., Steinitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastogenic+response+of+purified+human+T-lymphocyte+populations+to+Epstein-Barr+virus+(EBV).">Blastogenic response of purified human T-lymphocyte populations to Epstein-Barr virus (EBV).</a> Clin Exp Immunol. 30 (3), 347-353 (1977).</li><li>Sanderson, R. J., Rulon, K., Groeneboer, E. G., Talmage, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+murine+splenic+lymphocytes+to+concanavalin+A+and+to+co-stimulator.">The response of murine splenic lymphocytes to concanavalin A and to co-stimulator.</a> J Immunol. 124 (1), 207-214 (1980).</li><li>Decoursey, T. E., Chandy, K. G., Gupta, S., Cahalan, 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=Mitogen+induction+of+ion+channels+in+murine+T+lymphocytes.">Mitogen induction of ion channels in murine T lymphocytes.</a> J Gen Physiol. 89 (3), 405-420 (1987).</li><li>Nibbering, P. H., Zomerdijk, T. P., Tilburg, A. J., Furth, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+cell+volume+of+human+blood+leucocytes+and+resident+and+activated+murine+macrophages.">Mean cell volume of human blood leucocytes and resident and activated murine macrophages.</a> J Immunol Methods. 129 (1), 143-145 (1990).</li><li>Segel, G. B., Cokelet, G. R., Lichtman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+lymphocyte+volume:+importance+of+reference+particle+deformability+and+counting+solution+tonicity.">The measurement of lymphocyte volume: importance of reference particle deformability and counting solution tonicity.</a> Blood. 57 (5), 894-899 (1981).</li><li>Cooper, H. L., Braverman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+in+resting+and+growth-stimulated+human+peripheral+lymphocytes.+Evidence+for+regulation+by+a+non-messenger+RNA.">Protein synthesis in resting and growth-stimulated human peripheral lymphocytes. Evidence for regulation by a non-messenger RNA.</a> Exp Cell Res. 127 (2), 351-359 (1980).</li><li>Cooper, H. L., Braverman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+initiator+methionyl-tRNA+level+and+rate+of+protein+synthesis+during+human+lymphocyte+growth+cycle.">Close correlation between initiator methionyl-tRNA level and rate of protein synthesis during human lymphocyte growth cycle.</a> J Biol Chem. 256 (14), 7461-7467 (1981).</li><li>Teague, T. 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=Activation+changes+the+spectrum+but+not+the+diversity+of+genes+expressed+by+T+cells.">Activation changes the spectrum but not the diversity of genes expressed by T cells.</a> Proc Natl Acad Sci U S A. 96 (22), 12691-12696 (1999).</li><li>Tzur, A., Kafri, R., Lebleu, V. S., Lahav, G., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+and+size+homeostasis+in+proliferating+animal+cells.">Cell growth and size homeostasis in proliferating animal cells.</a> Science. 325 (5937), 167-171 (2009).</li><li>Messele, 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=Nonradioactive+techniques+for+measurement+of+in+vitro+T-cell+proliferation:+alternatives+to+the+[3H]thymidine+incorporation+assay.">Nonradioactive techniques for measurement of in vitro T-cell proliferation: alternatives to the [3H]thymidine incorporation assay.</a> Clin Diagn Lab Immunol. 7 (4), 687-692 (2000).</li><li>Maghni, K., Nicolescu, O. M., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitability+of+cell+metabolic+colorimetric+assays+for+assessment+of+CD4++T+cell+proliferation:+comparison+to+5-bromo-2-deoxyuridine+(BrdU)+ELISA.">Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA.</a> J Immunol Methods. 223 (2), 185-194 (1999).</li><li>Weichert, H., Blechschmidt, I., Schr&#246;der, S., Ambrosius, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MTT-assay+as+a+rapid+test+for+cell+proliferation+and+cell+killing:+application+to+human+peripheral+blood+lymphocytes+(PBL).">The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL).</a> Allerg Immunol (Leipz). 37 (3-4), 139-144 (1991).</li><li>Huang, K. T., Chen, Y. H., Walker, 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=Inaccuracies+in+MTS+assays:+major+distorting+effects+of+medium,+serum+albumin,+and+fatty+acids.">Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids.</a> Biotechniques. 37 (3), 410-412 (2004).</li><li>Rampersad, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+applications+of+Alamar+Blue+as+an+indicator+of+metabolic+function+and+cellular+health+in+cell+viability+bioassays.">Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays.</a> Sensors (Basel). 12 (9), 12347-12360 (2012).</li><li>Quent, V. M., Loessner, D., Friis, T., Reichert, J. C., Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrepancies+between+metabolic+activity+and+DNA+content+as+tool+to+assess+cell+proliferation+in+cancer+research.">Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research.</a> J Cell Mol Med. 14 (4), 1003-1013 (2010).</li><li>Augustine, N. H., Pasi, B. M., Hill, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ATP+production+in+whole+blood+and+lymphocyte+proliferation+in+response+to+phytohemagglutinin.">Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin.</a> J Clin Lab Anal. 21 (5), 265-270 (2007).</li><li>Sottong, P. R., Rosebrock, J. A., Britz, J. A., Kramer, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+T-lymphocyte+responses+in+whole-blood+cultures+using+newly+synthesized+DNA+and+ATP.">Measurement of T-lymphocyte responses in whole-blood cultures using newly synthesized DNA and ATP.</a> Clin Diagn Lab Immunol. 7 (2), 307-311 (2000).</li><li>Wallace, P. K., Muirhead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+2007:+a+proliferation+of+probes+and+applications.">Cell tracking 2007: a proliferation of probes and applications.</a> Immunol Invest. 36 (5-6), 527-561 (2007).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nat Protoc. 2 (9), 2049-2056 (2007).</li><li>Teague, T. K., Munn, L., Zygourakis, K., Mcintyre, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lymphocyte+activation+and+proliferation+by+video+microscopy+and+digital+imaging.">Analysis of lymphocyte activation and proliferation by video microscopy and digital imaging.</a> Cytometry. 14 (7), 772-782 (1993).</li><li>B&#246;hmer, R. M., Bandala-Sanchez, E., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+light+scatter+is+a+simple+measure+of+T-cell+activation+and+proliferation+but+is+not+universally+suited+for+doublet+discrimination.">Forward light scatter is a simple measure of T-cell activation and proliferation but is not universally suited for doublet discrimination.</a> Cytometry A. 79 (8), 646-652 (2011).</li><li>Lee, J., Sadelain, M., Brentjens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+murine+primary+T+lymphocytes.">Retroviral transduction of murine primary T lymphocytes.</a> Methods Mol Biol. 506, 83-96 (2009).</li><li>Gunzer, M., Weishaupt, C., Planelles, L., Grabbe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+negative+enrichment+of+CD4++and+CD8++T+cells+from+murine+spleen+via+nylon+wool+adherence+and+an+optimized+antibody+cocktail.">Two-step negative enrichment of CD4+ and CD8+ T cells from murine spleen via nylon wool adherence and an optimized antibody cocktail.</a> J Immunol Methods. 258 (1-2), 55-63 (2001).</li><li>Trickett, A., Kwan, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+stimulation+and+expansion+using+anti-CD3/CD28+beads.">T cell stimulation and expansion using anti-CD3/CD28 beads.</a> J Immunol Methods. 275 (1-2), 251-255 (2003).</li><li>P&#232;ne, J., Rahmoun, M., Temmerman, S., Yssel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+anti-CD3/CD28+mAb+coupled+magnetic+beads+permitting+subsequent+phenotypic+analysis+of+activated+human+T+cells+by+indirect+immunofluorescence.">Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence.</a> J Immunol Methods. 283 (1-2), 59-66 (2003).</li><li>Sigal, N. H., Dumont, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A,+FK-506,+and+rapamycin:+pharmacologic+probes+of+lymphocyte+signal+transduction.">Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction.</a> Annu Rev Immunol. 10 (1), 519-560 (1992).</li><li>Fruman, D. A., Klee, C. B., Bierer, B. E., Burakoff, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+phosphatase+activity+in+T+lymphocytes+is+inhibited+by+FK+506+and+cyclosporin+A.">Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.</a> Proc Natl Acad Sci U S A. 89 (9), 3686-3690 (1992).</li><li>Pollizzi, K. N., Waickman, A. T., Patel, C. H., Sun, I. H., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+size+as+a+means+of+tracking+mTOR+activity+and+cell+fate+of+CD4++T+Cells+upon+antigen+recognition.">Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T Cells upon antigen recognition.</a> PLoS One. 10 (4), e0121710 (2015).</li><li>Pollizzi, K. N., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cells+by+mTOR:+the+known+knowns+and+the+known+unknowns.">Regulation of T cells by mTOR: the known knowns and the known unknowns.</a> Trends Immunol. 36 (1), 13-20 (2015).</li><li>Mandala, 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=Alteration+of+lymphocyte+trafficking+by+sphingosine-1-phosphate+receptor+agonists.">Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.</a> Science. 296 (5566), 346-349 (2002).</li><li>Qin, X., 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=Sphingosine+and+FTY720+are+potent+inhibitors+of+the+transient+receptor+potential+melastatin+7+(TRPM7)+channels.">Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels.</a> Br J Pharmacol. 168 (6), 1294-1312 (2013).</li><li>Wulff, H., Kolski-Andreaco, A., Sankaranarayanan, A., Sabatier, J. M., Shakkottai, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulators+of+small-+and+intermediate-conductance+calcium-activated+potassium+channels+and+their+therapeutic+indications.">Modulators of small- and intermediate-conductance calcium-activated potassium channels and their therapeutic indications.</a> Curr Med Chem. 14 (13), 1437-1457 (2007).</li><li>Petho, Z., 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=The+anti-proliferative+effect+of+cation+channel+blockers+in+T+lymphocytes+depends+on+the+strength+of+mitogenic+stimulation.">The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation.</a> Immunol Lett. 171, 60-69 (2016).</li><li>Wulff, H., 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=Design+of+a+potent+and+selective+inhibitor+of+the+intermediate-conductance+Ca2+-activated+K++channel,+IKCa1:+a+potential+immunosuppressant.">Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.</a> Proc Natl Acad Sci USA. 97 (14), 8151-8156 (2000).</li><li>Lang, F., 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=Functional+significance+of+cell+volume+regulatory+mechanisms.">Functional significance of cell volume regulatory mechanisms.</a> Physiol Rev. 78 (1), 247-306 (1998).</li><li>Hue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+liver+carbohydrate+and+fatty+acid+metabolism+by+cell+volume.">Control of liver carbohydrate and fatty acid metabolism by cell volume.</a> Biochem Soc Trans. 22 (2), 505-508 (1994).</li><li>O'Flaherty, J. T., Kreutzer, D. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+aggregation+and+swelling+induced+by+chemotactic+agents.">Neutrophil aggregation and swelling induced by chemotactic agents.</a> J Immunol. 119 (1), 232-239 (1977).</li><li>Hsu, L. S., Becker, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+changes+induced+in+rabbit+polymorphonuclear+leukocytes+by+chemotactic+factor+and+cytochalasin+B.">Volume changes induced in rabbit polymorphonuclear leukocytes by chemotactic factor and cytochalasin B.</a> Am J Pathol. 81 (1), 1-14 (1975).</li><li>Rosengren, S., Henson, P. M., Worthen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration-associated+volume+changes+in+neutrophils+facilitate+the+migratory+process+in+vitro.">Migration-associated volume changes in neutrophils facilitate the migratory process in vitro.</a> Am J Physiol. 267, C1623-C1632 (1994).</li></ol>

The authors have no competing financial interests.

这里，我们描述了使用自动细胞计数器对T细胞母细胞转化的快速检测和定量的技术。根据我们的条件下（250毫微克/毫升PMA和250纳米离子霉素刺激），在细胞表面面积增加两倍和后活化的48小时的体积三倍。该测定是足够灵敏活化的前12个小时，其中，相对于静止（ 图2D）细胞体积增加只有1.25倍期间检测爆破。用抗CD3和抗CD28抗体包被的磁珠T细胞活化的多种生理上有关的机制产生了显著母细胞化反应，在体积平均2.3倍的增加（活化后72小时， 图3D）。人单核细胞表现出量在PMA / 2.6倍的变化离子霉素刺激48小时（ 图4）。该ICR小鼠脾脏T细胞平均直径和体积被确定为6.9微米，1.9×10 -4 NL，分别。人PBMC（从一个健康供体）具有7.7微米的平均直径以及2.7×10 -4 NL的平均体积。使用该协议，我们还测试增加PMA浓度他们为激活T细胞的能力。这些单细胞的结果与在类似的PMA浓度进行增殖实验一致。 据报道，影响细胞增殖的几种化合物进行了测试:31,32 FTY720;免疫抑制剂环孢素A，FK506，雷帕霉素27,28;和电车-34，钙激活KCa3.1通道33,35的阻断剂。我们发现，在测试的浓度下，胚细胞的最有效的抑制剂是环孢素和FK506。雷帕霉素对芽生一个较小但有统计学显著抑制作用。 TRAM-34，在另一方面，没有影响胚细胞。 环孢素A抑制既芽生和proliferation，但不能完全（ 图2B和2C），这表明自动细胞计数器测量是在T细胞增殖药物的效率良好的相关性。与环孢素一起雷帕霉素的存在下，胚被完全抑制，这表明在组合NFAT-和mTOR的介导的途径可以根据用PMA /伊屋诺霉素（ 图3C）促有丝分裂的刺激充分说明T细胞肿大。 一些增殖测定的动态范围是有限的（ 见图2）。增殖测定，如我们所使用的（ 图2C），报告说，包括来自凋亡和坏死细胞的贡献的信号。自动直径的变化的测量没有任何限制，因为该测量涉及仅活细胞。细胞活力用台盼蓝染色的健康，活细胞排除评估。在三围尺寸，使用前向在流式细胞仪双重细胞的辨别光散射是有问题的22。在自动细胞计数器测量，没有双峰人口被发现（ 图1）。另外，测量是足够精确100和200nM的环孢素（ 表1）和促有丝分裂刺激12小时（ 图2D）内检测芽生的效果来区分。 这个新的测定法是对样品的小号码是特别有用的。多达15个样品可以在1小时内进行测量。然而，该测定也有其局限性，其中大部分可以缓解。即使它可以有效地在气泡径解决小（&lt;1微米）的差异，我们使用了机器模型具有5μm的更低的检测阈值。此限制可能会导致非常小的单元尺寸的高估，因为它有效地排除了所有的细胞直径较小。然而，细胞计数的较新的机型检测误差阈值〜2微米的孩子，这应该缓解此问题。如果有太多的碎片样品中，该仪器将它们视为活细胞。此外，气泡可以偶尔进入流动池，并导致由机器捕获的细胞的图像的失真。失真似乎不影响生存力测定，但它可能会影响测量的直径。因此，建议所有的图像被实验者为气泡来检查从每个试验的数据被接受分析前。由于软件仅绘制围绕细胞圈，直径非球形细胞可以是朝向长轴偏斜，使得该测定不太适于非球形细胞。 该测定法可快速，取每个样品约4分钟相比，增殖测定，这需要几个小时。数据收集也相当简单使用设备附带的软件。该软件可以将测量数据导出到SPreadsheet进行分析。最后，这是一种单细胞，而不是一个人口，测量;这两个芽生和扩散测量;它同时可行和死细胞区分开来。它可以用于其安装的免疫应答的能力评价各种小鼠品系。该测定也可以成功地用于芽生中从人类供体（ 图4）的T细胞的测量，而且它也可以在其它细胞类型中。 细胞体积增大已知有助于代谢的调节:细胞肿胀促进肝细胞36,37谷氨酰胺诱导肝糖合成和脂肪形成。中性粒细胞显示35-60％的迁移相关的体积增大，而趋化剂诱导10-15％的肿胀38-40。电流测定可以潜在地用于研究这些进程。

T淋巴细胞是负责在哺乳动物适应性免疫原代细胞。已知的是它们由MHC分子的抗原呈递细胞的表面上呈现的特定抗原肽反应。在同源的T细胞受体（TCR）的活化，所述细胞扩大的过程称为母细胞转化，或胚。施加1刺激之后这个过程是在第一〜6小时检测的。在芽生，个别T细胞的数量增加2〜4倍，2-6。淋巴细胞开始在一个称为克隆扩增过程增殖，其目的是产生抗原特异性TCR的承载细胞尽可能许多克隆。然后，子代细胞分化成细胞毒性（CD8 +）或辅助（CD4 +）效应T细胞发挥其免疫功能。因此，在人或小鼠血幼稚T淋巴细胞是在细胞的G- 0（静止）期周期和支持最少的代谢活动。在暴露于抗原或有丝分裂原，T细胞重新进入细胞周期，具有转录和蛋白质合成7-10的伴随刺激。有丝分裂原，如佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），并通过蛋白激酶C（PKC）和Ca 2+依赖性信号传导途径1的激活离子霉素刺激的淋巴细胞。 T细胞用PMA /伊屋诺霉素的活化绕过TCR信令步骤。 体外增殖测定法广泛用于评估淋巴细胞功能和响应于刺激的目的。增殖读数通常采取的T细胞刺激开始后一至三天，并反映数百或数千细胞的集体状态。各种促细胞分裂剂和免疫调节药物在体外的效力可以通过简单地测量在这些化合物的存在下增殖率进行评价。有些一中ssays及其限制在下面讨论。 对于直接的细胞数计数，该过程是耗时的，与操作者的错误的概率高。 DNA合成，所述3 H-胸苷掺入法测量DNA的合成，但其主要限制是它的放射毒性。非放射性的替代方法是BrdU的，但对于细胞生长的线性响应的范围是有限的，抗体治疗是必需的，从而增加了该过程11,12的步骤的数量。 用于代谢活性，四唑盐（MTT，MTS，XTT，和WST-1）和刃天青染料基比色法报告分裂细胞群体的总体代谢状态。然而，MTT不溶于培养基中，需要额外的洗涤步骤，因而掺入的测量误差; XTT需要额外的组件，以有效降低; MTS-，WST-1，和基于刃天青，测量影响通过培养基的pH及其组件血清，白蛋白或酚红13-16编这些测定法不测量存活细胞的实际数量，而是估计结合酶活性。因此，扩散速率可能不能准确地由因为细胞数和染料还原12,17非线性相关的代谢试验测定。 用于测定ATP浓度，在ATP的T细胞活化诱导的增加相关联以增殖。然而，细胞内ATP的高程是T细胞活化的初始步骤之一;很多步骤的背后，是实际的扩散17,18。 染料稀释法，CFSE荧光染料共价结合到细胞内蛋白质染色细胞。染料显示了荧光强度的增殖依赖性降低，这可以跟踪细胞分裂的次数。然而，由于共价蛋白标记，所述的这些功能蛋白质可受到损害。染料是对细胞有毒在较高浓度。在较低的染料浓度，然而，最初的荧光强度降低，减小了可被跟踪的细胞分裂的次数。此外，用CFSE标记后，有在第一个24至48小时的时间内，这限制了该测定19,20的动态范围初始荧光的增殖无关的〜50％的损失。 大多数这些测定的反映大量的细胞的集体状态，并且需要用荧光染料的细胞的治疗。坏死和凋亡细胞也可能有助于这些测量，除非它们从分析通过用化学药品或抗体染色除去。 淋巴细胞芽生可以通过各种方法，如光学显微镜进行评价或流式细胞4,21,22。在这里，我们描述了一种快速的方法为T细胞大小的使用测量Ñ自动细胞计数器，它收集存储并在稍后的时间可以被重新分析的实时细胞图像。除了大小的测量，该装置提供了精确的细胞数和存活细胞的百分比，如通过锥虫蓝染色排除法测定。在这个协议中使用的设备是可商购的，并且制造商测试使用三种不同的仪器和几个浓度和活力控制仪器的精确度。这些研究的结果证明方差的系数，这是一般低于6％。在协议中所指出的，该装置被校准定期与6微米和8微米直径的聚苯乙烯珠。使用细胞计数器基于小区直径静止的T细胞和T淋巴母细胞之间进行区分的优点是使用的易用性和分析的自动化性质。该软件能够绘制每个小区绕了一圈，并计算出细胞的直径。此外，即时年龄是可见的操作者，谁可以验证在识别细胞和正确地绘制他们周围的圆的仪器的精度。在限制方面，仪器不能本身碎片和细胞之间的区别;因此，重要的是在操作者观察的每个图像，因为它正在处理中。有用于掺入气泡，这将降低用于分析可用的字段数的电位;但是，如果执行定期冲洗保养，这是罕见的。 在这项研究中，脾T淋巴细胞的组，离子霉素和增加的PMA浓度为12-48小时刺激。 PMA浓度低至2毫微克/毫升诱导既稳健母细胞化反应和显著增殖。的几种药物，如免疫抑制剂环孢菌素A（CSA），FK506（他克莫司），和雷帕霉素（西罗莫司），以及离子通道阻滞剂TRAM-34和FTY720的影响的测量（fingoliMOD），在芽生表现出与增殖报告的影响基本一致。人PBMC与抗CD3和抗CD28抗体包被的磁珠PMA /离子霉素和小鼠T细胞刺激的母细胞化反应进行了测定。 细胞计数器测定量化既胚和增殖率（细胞密度）同时但分别不同于上述的方法，这看这些效应的组合。所提出的协议提供了一种评价有丝分裂和免疫调节剂的效力一个快速和强大的技术。

<table><tbody><tr><td>RPMI-1640&amp;nbsp;</td><td>Lonza</td><td>BW12-702F</td><td></td></tr><tr><td>40 &amp;mu;m nylon cell strainers&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>nylon wool fiber columns</td><td>Polysciences, Inc.&amp;nbsp;</td><td>21759-1</td><td></td></tr><tr><td>50 ml conical tubes&amp;nbsp;</td><td>The Lab Depot</td><td>TLD431697</td><td></td></tr><tr><td>6-well cell culture treated plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>96-well cell culture treated plates</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher BioReagents</td><td>BP231-100</td><td></td></tr><tr><td>CellTiter 96 AQueous One Solution Cell Proliferation Assay&amp;nbsp;</td><td>Promega</td><td>G3582</td><td>MTS based assay</td></tr><tr><td>Cyclosporine A</td><td>Sigma-Aldrich</td><td>30024</td><td></td></tr><tr><td>FK506</td><td>Cayman Chemical Company</td><td>104987-11-3</td><td></td></tr><tr><td>Rapamycin</td><td>Santa Cruz Biotechnology</td><td>sc-3504</td><td></td></tr><tr><td>TRAM-34</td><td>Sigma-Aldrich</td><td>T6700</td><td></td></tr><tr><td>FTY720</td><td>Sigma-Aldrich</td><td>SML0700</td><td></td></tr><tr><td>Dynabeads Mouse T-Activator CD3/CD28</td><td>Gibco</td><td>11456D</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)&amp;nbsp;</td><td>Acros Organics&amp;nbsp;</td><td>356150010</td><td></td></tr><tr><td>Ionomycin calcium salt</td><td>Sigma-Aldrich</td><td>I0634</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>MP Biomedicals&amp;nbsp;</td><td>ICN1670049</td><td>100x stock</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>HyClone</td><td>SH30378.02</td><td>10x stock</td></tr><tr><td>1,4-dithiothreitol (DTT)&amp;nbsp;</td><td>Research Products International</td><td>12/3/3483</td><td>reducing agent</td></tr><tr><td>8 &amp;micro;m micro particle size standard&amp;nbsp;</td><td>Sigma-Aldrich</td><td>84192-5ML-F</td><td>Actual&amp;nbsp; 8.02 &amp;micro;m</td></tr><tr><td>6 &amp;micro;m micro particle size standard&amp;nbsp;</td><td>Sigma-Aldrich</td><td>89756-5ML-F</td><td>Actual 6.084 &amp;micro;m</td></tr><tr><td>Single magnetic separation stand for 1.5 - 2 ml tube</td><td>V&amp;amp;P Scientific, Inc.</td><td>VP772F5</td><td></td></tr><tr><td>Cell culture incubator</td><td>Forma Scientific&amp;nbsp;</td><td>3110</td><td></td></tr><tr><td>Synergy H1 hybrid reader&amp;nbsp;</td><td>Bio Tek</td><td>BTH1M</td><td></td></tr><tr><td>Vi-CELL cell viability analyzer&amp;nbsp;</td><td>Beckman Coulter</td><td>731050</td><td></td></tr></tbody></table>

rapid quantification of mitogen-induced blastogenesis in t lymphocytes for identifying immunomodulatory drugs

响应淋巴细胞增殖对抗原或有丝分裂的刺激是检验免疫调节（ 即免疫抑制或免疫）化合物和生物制剂有用容易量化的现象。其中的有丝分裂过程中最早的步骤是细胞肿大或母细胞转化，于是分割前的细胞体积增大。它通常是在第一几个小时的T淋巴细胞刺激可检测的。在这里，我们描述了一种快速方法，使用一个自动细胞计数器来量化在从小鼠脾和人外周血单核细胞（PBMC）分离的T淋巴细胞芽生。在大多数情况下各种常用增殖测定是费力的并且仅反映了总体人口的影响，而不是一个种群中的单个细胞的影响。与此相反，所提出的自动细胞计数器测定规定的泡孔直径，可以迅速，直接和精确的测量用于评估各种促细胞分裂剂和免疫调节药物在体外的效力。

我们使用该测定用PMA，佛波酯和离子霉素，钙离子载体刺激期间比较淋巴细胞爆炸形成。 图1A示出休息的直径上和药理学激活脾T细胞的频数分布。用PMA /离子霉素为〜2天细胞治疗导致在朝向更大的直径（例如参见参考文献4）的分布的中值一个显著转变。直径较小的细胞的数目也相应减少。为了确定我们的设备报告正确的直径，我们进行了两次校准为6微米的聚苯乙烯珠和8微米的直径（参见材料 数据表 ）。 图1B和1C示出的读数从叠加有重叠有6微米的标准8微米标准和静息T细胞活化的T细胞。数字图1D示出了由制造商报告的控制珠粒尺寸作图与我们的自动细胞计数器测定的中值直径。它似乎在6微米大小是在我们的测量结果稍有高估，可能是由于具有5微米的下限的仪器的测量阈值。然而，从细胞计数器测量计算出的胎圈直径的标准偏差为在与由制造商报告的标准偏差的协议。我们从这些实验，该装置能够可靠地用于比较休息和丝裂原刺激小鼠T细胞的大小和该装置能准确地测量电池实际直径结束。 然后我们进行测试位于PMA浓度的平均直径增加的依赖性，结果发现，在250纳米的固定洛诺霉素浓度，对PMA效果不显著通过提高其浓度˚F改​​变ROM 2至250毫微克/毫升（ 图2A）。使用基于MTS测定法，我们测量T细胞增殖，在50和250毫微克/毫升的PMA，并没有发现明显的差异（ 图2C），在协议与我们的气泡直径的数据。钙调磷酸酶抑制剂药物环孢素A（300纳米）和FK506（1纳米）抑制两胚（ 图2B）和增殖（ 图2C）。抑制未在这两种情况下完成的，但是。测量进行12小时后的PMA /离子霉素加成显示在直径增加为50纳克/毫升和250纳克/毫升的PMA，分别（ 图2D）7.2％和6.8％。在这两种浓度大小的增加并不显著不同，因为是48小时刺激（比较图2A）的情况。 后PMA /离子霉素刺激48小时从静息收集的气泡直径的数据和活化的T细胞中总结了<strong&gt;的表1。搁ICR小鼠脾T细胞的平均表面面积和体积分别为151.4微米2和1.9×10 -4 NL，分别。活化的T细胞的平均表面积和体积分别为300.6微米2和5.9×10 -4 NL，分别。对于所有激活的细胞中的总平均直径增加是40.92％（ 表1）。 我们还测试报告是免疫抑制其它化合物:环孢素，他克莫司，雷帕霉素，FTY720和电车-34。在图3中 ，在静息获得单元尺寸的测量和活化的T细胞在不存在和这些药物的存在下被示出。环孢素A和FK506，靶向活化T细胞（NFAT）27,28的钙调神经磷酸依赖性核因子，是最有效的，抑制由近72％（ 图 3A和3B）的母细胞化反应。有趣的是，与增加PMA浓度，CSA和FK506的效力有所回落，即使有在没有这些药物（ 图2A和2B）没有额外的细胞直径增大。雷帕霉素，抑制雷帕霉素（mTOR的）29,30的哺乳动物靶点的免疫抑制剂，具有适中但显显著影响（ 图3A，3B和3C）。 FTY720是鞘氨醇1-磷酸受体激动剂抑制淋巴细胞出口进入流通领域31，最近发现抑制TRPM7，T中高度表达淋巴细胞32阳离子通道。既FTY720和雷帕霉素对芽生可比的效果，从平均直径增加52％的减少它至约34％（ 图3A和3B）。 TRAM-34是钙激活钾通道KCa3.1 33的阻断剂。在700nm的测试，TRAM-34在小鼠T细胞无效，我ñ按照最近的人类T细胞增殖的研究;其效力可取决于分裂素刺激34（ 图3A和3B）的性质和强度。 700纳米TRAM-34是有效的，在我们的膜片钳电生理学实验阻断KCa3.1通道（未示出数据）。这种比较是静止的和药物暴露的细胞之间在多个试验在48小时由相同实验内。当雷帕霉素和环孢素被一起使用时，胚被完全抑制（ 图3C）。 鼠T细胞用抗CD3 /抗CD28包被的72小时磁珠活化产生的平均直径，这是减少到5％环孢素的存在（ 图3D）增加27％。在48小时的PMA /离子霉素活化的人类单核细胞由33％的直径增加，并且活化环孢素的存在下反应降低到23％（ <strong&gt;的图4）。 <img alt="图1" src="/files/ftp_upload/55212/55212fig1.jpg" /> 图1: 小鼠脾脏T细胞直径的频率分布。 （A）黑色列表示休息和红柱表示PMA /离子霉素激活（48小时）细胞。叠加在8微米的颗粒大小的校准标准脾激活T细胞（B）分布。 （ 三 ）休息叠加到6微米的校准标准T细胞的分布。使用的细胞和珠的数量在盒子表示。 （D）的平均通过细胞计数器测定直径作图由制造商报告的珠径。 <a href="http://ecsource.jove.com/files/ftp_upload/55212/55212fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ="1"&gt; - 页面内<img alt="图2" src="/files/ftp_upload/55212/55212fig2.jpg" /> 图2: 在PMA浓度的鼠T细胞活化的依赖性。 （A）T细胞未处理或2，50治疗，250毫微克/毫升PMA在固定霉素浓度（250纳米）的平均直径。静止的（B）的细胞计数器测量和激活（2，50，250纳克/毫升的PMA）T细胞在不存在和300nM的环孢素或1nM的FK506的存在。 （℃）的MTS在50和250纳克增殖测定/ ml的PMA与在不存在250纳米离子霉素和300nM的环孢素的存在。显著差异（p &lt;0.001），以表示星号。 n是试验的总数。数据是从3只小鼠分离的细胞。 （D）休息细胞直径和活化的T细胞（250毫微克/毫升的PMA和250纳米离子霉素）在没有和300nM的环孢素A的存在激活后12小时。<html> PLOAD / 55212 / 55212fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55212/55212fig3.jpg" /> 图3: 环孢素A的影响，FK506，FTY720，雷帕霉素，和对细胞大小TRAM-34。 （A和C）休息和PMA /离子霉素激活小鼠T细胞在没有和在场的药物平均直径。浓度示于框中。 （B）中从A数据表示为在静息T细胞的直径增加的百分比。与250毫微克/毫升的PMA和250牛霉素进行淋巴细胞活化，和48小时后取测量。 （D）的休息和抗CD3的细胞计数器测量/ CD28激活的T细胞，在不存在和300nM的环孢素的存在下活化后72小时。rge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55212/55212fig4.jpg" /> 图4: 在胚细胞有丝分裂时刺激人PBMC。 （A）黑色列表示休息和红柱表示激活的PBMC。 （B）的静止的平均直径和在不存在激活的PBMC和300nM的环孢素的存在。细胞用250纳克/毫升的PMA和250纳米离子霉素激活，激活后的48小时采取的测量。 <a href="http://ecsource.jove.com/files/ftp_upload/55212/55212fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> COLUMN1 </td><td>休息</td><td>活性</td><td>环孢素A 100纳米</td><td>环孢素A 200纳米</td></tr><tr><td>平均直径</td><td> 6.94微米</td><td> 9.78微米</td><td> 8.19微米</td><td> 7.92微米</td></tr><tr><td>在直径的平均增幅</td><td></td><td> 40.92％ </td><td> 17.88％ </td><td> 14.09％ </td></tr><tr><td>平均面积</td><td> 151.37平方微米</td><td> 300.61平方微米</td><td> 210.56平方微米</td><td> 197.22平方微米</td></tr><tr><td>表面积的平均增幅</td><td></td><td> 98.59％ </td><td> 39.00％ </td><td> 30.16％ </td></tr></tbody></table> 表1: 平均泡孔直径和表面积相对增加的综述。测量进行250毫微克/毫升的PMA和250纳米离子霉素激活后的48小时。

Watch this Scientific Journal Video about Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs at JoVE.com

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

rapid-quantification-mitogen-induced-blastogenesis-t-lymphocytes-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

18.7K 次观看.  Wright State University. T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters. 

视频: Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

科研

癌症研究

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

的IFN-γ酶联免疫斑点检测，以评估水痘 - 带状疱疹病毒特异性细胞免疫继脐血移植的发展

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

免疫与感染

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

医学

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

一种简单有效的方法，用于测试从人 CD14+ 单核细胞生成致耐受性树突状细胞的免疫调节剂

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

测量初始和效应 CD8 T 细胞的线粒体功能

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

Source: Fouda, A., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55199">A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules.</a> J. Vis. Exp., (2017).
This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds&#39; stimulatory and inhibitory effects, respectively.

使用工程淋巴细胞筛选免疫调节化合物的基于荧光的测定

1. Preparation of Splenocyte Medium and Flow-cytometry Buffer

	Perform all steps under a 70% ethanol-cleaned biological hood.
	Remove 70 mL of pre-warmed ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

一种监测佐剂介导的细胞毒性 T 淋巴细胞生成的免疫学技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

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