笔者想在雷诺实验室的医学和科学的罗莎琳德·富兰克林大学的所有成员，并在得克萨斯大学MD安德森癌症中心大学的陈岽实验室感谢这个协议的优化。这项工作得到了一笔赠款，JMR来自美国国立卫生研究院（K22AI104941）的支持。

所有的实验程序都采用经环境健康和安全的医学和科学的罗莎琳德·富兰克林大学办公室协议执行。用于该协议C57BL / 6小鼠（来自NCI购买）被安置无特定病原体的条件下，所有的动物实验，使用在医学罗莎琳德富兰克林大学批准的机构动物护理和使用委员会（IACUC）的协议执行，并且科学。 1.准备仪器，耗材和试剂的<ol><li>在无菌PBS，覆盖膜，封口膜中的大衣48孔或24孔板抗CD3和抗CD28（ 表1），以防止蒸发和污染，然后孵化O / N在4（C T）前一天细胞分离。另外，外套1井在37℃。1H通过在0.5-1毫升涂敷既抗CD3和抗CD28在1微克/ ml至执行的Th0，Th1细胞，Th2细胞，和iTreg条件TAL体积（48孔板）。对于Th17细胞时，我们通常涂层抗CD3在2微克/毫升和抗CD28在1微克/毫升。 注意:我们已发现，较低的抗CD3浓度是最适合的Th1，Th2细胞，和iTreg生成而较高的抗CD3浓度是最佳的Th17细胞产生。因此，抗CD3和抗CD28的浓度应该滴定和单个实验室（ 表2）进行了优化。 </li><li>通过高压灭菌或消毒液消毒一套清扫工具（剪刀和镊子）。管或含70％乙醇的小烧杯中，将需要用于解剖的工具快速杀菌作用。最后，将需要解剖前灭菌鼠标的表面含有70％乙醇的喷射瓶。 </li><li>通过包装一块平坦的泡沫塑料或软木板铝箔准备剥离面。解剖销或针用于固定到位的动物。 </li><li>准备菜肴或板为TI的集合ssues。如果集中组织，独立60毫米菜含3毫升无菌PBS + 1％FBS（P​​BS +）的脾和淋巴结组织就足够了。如果从单个小鼠保持组织中分离出来，制备PBS +的含24孔板1毫升在井的所需数目。 </li><li>在使用前制备的完全RPMI培养基进行细胞培养和预温热（ 表1）。 注意:我们经常使用的RPMI但其他媒体，例如IMDM和DMEM可以是同等有效的或优于根据实验室和实验。 </li><li>切的2x2厘米尼龙的正方形网眼带120微米孔径和高压釜灭菌（ 表1）。 </li><li>可选:如果使用高速磁细胞分选系统，如autoMACS对CD4 +细胞的富集，预暖运转和漂洗缓冲液在37℃的水浴，以防止损坏机器。 </li><li>注:富集建议提高分拣产量和减少排序时间。 </li><li>所有准备步骤，除了夹层，应当在无菌组织培养罩进行。 </li></ol>从小鼠2.隔离淋巴结和脾脏的<ol><li>安乐死使用体制批准的技术动物的所需数量。使用C57BL / 6雌性小鼠，年龄5-10周，由于幼稚T细胞和下半身脂肪相比，男性比例高或年纪较大的老鼠。 注:从雄性小鼠衍生的细胞将在体外进行同样的协议步骤保持相同。雌性小鼠，5-10周龄，典型地将产生3-6×10 6个幼稚每只小鼠的CD4 + T细胞。然而，使用不同的小鼠品系可能会影响成品率和性能。例如，从C57BL / 6小鼠细胞产生Th1细胞而从Balb.c小鼠细胞是用于产生Th2细胞更好更好。 </li><li>传播动物的四肢和如图1引脚他们的地方。切开皮肤长itudinally从肛门到下巴，注意不要穿刺深层组织。 </li><li>蔓延开钳皮肤和PIN码的地方，以方便进入淋巴结皮肤， 如图1。 </li><li>从图1中所示的位置收获的可访问的淋巴结。抓取的淋巴结有一对镊子的同时与另一对钳子的分离组织将允许容易地移除。放置在收集盘组织。 </li><li>收获从图1所示位置的脾脏。在这种情况下，仔细切割进入腹膜是需要获得脾脏。将集合中菜的组织。 </li><li>重复此过程，如果更多的组织会有所收获。否则，继续下一步骤。在这一点上，执行所有剩余的步骤在冰上，直到T细胞的分选后的电镀。 </li></ol> 3，组织处理和CD4 +富集<ol><li>处理淋巴结和脾脏在不同的菜，以增加产率，为淋巴结细胞制剂不需要红细胞裂解，这可能导致轻微白细胞死亡。因此，下面的步骤描述了用于处理脾和淋巴结群体分别随后进行排序之前结合到标签的方法。 </li><li>删除收集的菜和地点组织（脾或合并淋巴结）中含有3毫升的PBS +的全新60毫米的菜。放置无菌尼龙的平方用无菌镊子啮合在组织。 </li><li>以一个注射器活塞的拇指侧（3-10毫升），并轻轻地粉碎对网格组织。几乎所有的组织应该进入暂停。另外，将组织2蒸压磨砂载玻片之间研磨成混悬液。 </li><li>吸取悬挂上下几次分手其余的可溶性团块。将新方片尼龙网超过一开幕的15毫​​升锥形管中，并通过过滤悬浮液，以除去剩余的碎屑。 </li><li>额外的淋巴结和脾脏组织重复步骤2-4。 </li><li>一旦悬浮液被过滤到15ml的管中填充用PBS +上的管的其余部分并倒转几次。离心细胞在475 XG 5分钟，在4℃ </li><li>对于脾细胞，吸出上清液离心后，重悬在1ml的每脾冰冷1X ACK溶液将细胞沉淀1分钟以裂解红细胞。然后加入10毫升的PBS +对所述ACK的顶端，倒置的管，和离心机475×g离心5分钟，在4℃。 </li><li>为淋巴结细胞，吸在PBS + 2毫升上清液和重悬。如果需要的话，这些可以与他们的ACK裂解和洗涤步骤后的脾细胞进行组合。 </li><li>吸出的脾细胞离心，重悬在另外10毫升的PBS +后，上清。在这一点上的淋巴结悬浮可以是一个dded对脾胃停牌前是否需要进行分类组织池。再次离心管中，在475 XG为5分钟，在4℃。 </li><li>将细胞沉淀，现在可以对CD4富集制备。如果此步骤将不会被执行，则继续执行分拣准备步骤。 </li><li>对于使用高速磁性细胞分选系统的CD4富集，重悬与CD4珠的细胞团。通常为15微升珠子稀释在85微升PBS +的用于从1小鼠标记组织。根据需要坡道珠混合物的体积，悬浮颗粒，孵育15-30分钟，在4℃ </li><li>用10毫升PBS +洗细胞，通过尼龙悬挂到一个新的15ml试管中以4℃再次清除杂物，并离心475 XG 5分钟</li><li>重悬在100μl每只小鼠PBS +的沉淀并进行正选择高速磁性细胞分选系统。如果使用的是手动分离系统中，follow制造商的说明富集。可替代地，重悬在FACS缓冲液中的样品:PBS中1mM的EDTA（pH值= 8）和1％BSA，无菌过滤。 </li><li>取出阳性分数，并再次洗涤，用PBS + 10毫升。富集的CD4 +分数现在已经准备好被标记进行排序。 </li></ol> 4.排序朴素CD4 + T细胞<ol><li>标记的细胞沉淀用PBS +针对CD62L，CD44，CD25，和CD4含有荧光抗体。如果使用表1中所列的抗体，提供稀释的每个。用于染色的抗体混合物的100μl的体积是从1小鼠用于每个组织。 </li><li>悬浮在抗体的鸡尾酒颗粒，并在黑暗中孵育15-30分钟，在4 摄氏度 。 </li><li>与PBS + 10毫升和离心机475 XG为5分钟，在4 摄氏度洗细胞</li><li>在另一尼龙过滤器或电池滤网盖通过混合重移动任何剩余的碎片。吸取标记的细胞成管是用于细胞分选仪相兼容。保持细胞在冰上，并从光直到排序的保护。 </li><li>通过取适量涂于脸部完全RPMI培养基或胎牛血清的吹打插入细胞分选仪管集合兼容的底准备收集管。 </li><li>排序幼稚CD4 + T细胞的CD4 + CD25 - CD62L + CD44 -群（ 图2）。用完全RPMI培养基洗涤收集的细胞，并离心，如上所述。 </li><li>重悬在完全RPMI粒料，计数的幼稚细胞中，并调节浓度至1×10 6个细胞/ ml。 </li></ol> 5.设置在体外分化<ol><li>吸出抗CD3和抗CD28包被的板的孔中，并用无菌PBS（无FBS）的1ml中井洗每个。对于48孔板，板0.5×10 6个细胞/孔在0.5的RP毫升MI。对于24孔板，培养1×10 6个细胞/孔在1.0的RPMI的溶液中。 </li><li>如果任一之前，测试的一个因素的影响，例如药物加该物质到适当的孔中，或根据实验分化过程后。接着，补充培养基与细胞因子和阻断抗体相应。 TH0:30 U / ml的人IL-2。 TH1:15毫微克/毫升的小鼠白介素12，30 U / ml的人IL-2，和5000毫微克/毫升11B11.Th2:10毫微克/毫升的小鼠白介素4，30 U / ml的人IL-2，2000纳克/毫升的可溶性37.51，和5000纳克/毫升XMG1.2。 iTreg:15毫微克/毫升hTGFβ，30 U / ml的人IL-2，5000纳克/毫升XMG1.2和5000纳克/毫升11B11。 TH17:20毫微克/毫升小鼠白介素6，3毫微克/毫升hTGFβ，5000纳克/毫升XMG1.2和5000纳克/毫升11B11。 请注意:上述的条件被认为是最适合作为在分化实验（ 代表结果部分）的末端测量最大化细胞因子的产生。但是，各条件应该为各个实验室（ 表2被优化</stroNG&gt;）。此外，除了与板结合的抗CD28加入可溶性抗CD28已经发现，以提高在我们的实验室的Th2细胞因子的产生，但对其它的T细胞亚群没有影响: </li><li>孵育板在37℃下用5％CO 2的4-5之前的基因表达和细胞因子产生的分析ð。可替代地，取出从TCR刺激细胞后2天，调整为1×10 6个细胞/ ml用新鲜培养基，并在板新井（非涂布）来增强细胞增殖和最终产率。在这种情况下，是不需要的新鲜偏振细胞因子和抗体，但IL-2的10单位/毫升应添加到新媒体的Th0，Th1细胞，Th2细胞，和iTreg培养物。 </li></ol>分化6.分析<ol><li>对于通过流式细胞术细胞因子分析，用10ng / mL的PMA和1000以及重新刺激每纳克/ ml的离子霉素或1微克的高尔基抑制剂的存在下，例如布雷菲德菌素A / ml的抗CD3 4-6小时（ 表1）。执行内细胞因子染色取决于实验（ 图3）。 注:色漆非谱系特异性细胞因子或转录因子，如IL-4（未示出）和IFNγ（ 图3），用于Th17细胞培养物，对每一个条件作为阴性对照，并作为分化效率的指标。此外，染色的Foxp3对于Th17细胞培养物作为调节性T细胞的倒数发展可与加成TGFβ的发生。 </li><li>用于蛋白质定量，通过细胞因子特异性ELISA（ 图3）收集来自各孔测定上清液。如果有令人担忧的是被测试的因子已经影响增殖相比对照样品，所述细胞可洗涤，计数，归一化，然后用1μg/ ml的抗CD3的24小时再刺激收集上清用于ELISA分析之前。如果测定的IL-4从Th2细胞的条件下，清洗和再刺激必须除去的IL-4，其可以是升eftover从最初分化培养。 </li><li>对于通过实时PCR的基因表达分析，收获细胞的潜伏期后，洗净，正常化，并为2-6ħ重新刺激用抗CD3的1微克/毫升之前，收获的mRNA（ 图3）。 </li></ol>

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E., 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=Interleukin+17-producing+CD4++effector+T+cells+develop+via+a+lineage+distinct+from+the+T+helper+type+1+and+2+lineages.">Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages.</a> Nat Immunol. 6 (11), 1123-1132 (2005).</li><li>Langrish, C. 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=IL-23+drives+a+pathogenic+T+cell+population+that+induces+autoimmune+inflammation.">IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.</a> J Exp Med. 201 (2), 233-240 (2005).</li><li>Park, 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=A+distinct+lineage+of+CD4+T+cells+regulates+tissue+inflammation+by+producing+interleukin+17.">A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.</a> Nat Immunol. 6 (11), 1133-1141 (2005).</li><li>Ivanov, I. I., 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+orphan+nuclear+receptor+RORgammat+directs+the+differentiation+program+of+proinflammatory+IL-17++T+helper+cells.">The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126 (6), 1121-1133 (2006).</li><li>Chung, Y., 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=Critical+regulation+of+early+Th17+cell+differentiation+by+interleukin-1+signaling.">Critical regulation of early Th17 cell differentiation by interleukin-1 signaling.</a> Immunity. 30 (4), 576-587 (2009).</li><li>Reynolds, J. M., Martinez, G. J., Chung, Y., Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+signaling+in+T+cells+promotes+autoimmune+inflammation.">Toll-like receptor 4 signaling in T cells promotes autoimmune inflammation.</a> Proc Natl Acad Sci U S A. 109 (32), 13064-13069 (2012).</li><li>Reynolds, J. M., 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=Toll-like+receptor+2+signaling+in+CD4(+)+T+lymphocytes+promotes+T+helper+17+responses+and+regulates+the+pathogenesis+of+autoimmune+disease.">Toll-like receptor 2 signaling in CD4(+) T lymphocytes promotes T helper 17 responses and regulates the pathogenesis of autoimmune disease.</a> Immunity. 32 (5), 692-702 (2010).</li><li>Wu, 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=Induction+of+pathogenic+TH17+cells+by+inducible+salt-sensing+kinase+SGK1.">Induction of pathogenic TH17 cells by inducible salt-sensing kinase SGK1.</a> Nature. 496 (7446), 513-517 (2013).</li><li>Ghoreschi, 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=Generation+of+pathogenic+T(H)17+cells+in+the+absence+of+TGF-beta+signalling.">Generation of pathogenic T(H)17 cells in the absence of TGF-beta signalling.</a> Nature. 467 (7318), 967-971 (2010).</li><li>Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+the+context+of+an+inflammatory+cytokine+milieu+supports+de+novo+differentiation+of+IL-17-producing+T+cells.">TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.</a> Immunity. 24 (2), 179-189 (2006).</li><li>Avni, O., 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=T(H)+cell+differentiation+is+accompanied+by+dynamic+changes+in+histone+acetylation+of+cytokine+genes.">T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes.</a> Nat Immunol. 3 (7), 643-651 (2002).</li><li>Matheu, M. P., 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=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), (2007).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+th17+differentiation+of+naive+CD4+T+lymphocytes.">Isolation and th17 differentiation of naive CD4 T lymphocytes.</a> J Vis Exp. (79), e50765 (2013).</li></ol>

作者宣称没有竞争的财务权益。

而脾包含幼稚Th细胞，这部分人群在淋巴结的比例要高得多。未能正确识别和清除淋巴结此协议将导致幼稚细胞的一个贫穷的产量。这可以在具有更多的脂肪组织老年小鼠或雄性小鼠尤其困难。 如图动物四肢和皮肤的1，适当的固定和钉扎将允许访问的外部淋巴结容易可视化。一旦淋巴结和脾脏中进行处理，细胞分选是得到高度纯化的幼稚CD4 + T细胞群体的首选方?...

不同的谱系或CD4 + T辅助细胞亚群的概念（TH）细胞已自20 世纪1的后半部被周围。在的共刺激信号的结果存在关联抗原的几轮的细胞增殖和最终分化成效应Th细胞识别。在此过程中产生的Th细胞的类型取决于活化2中存在的细胞因子环境。最初，幼稚Th细胞被认为极化到下面的T细胞受体（TCR）的活化，共刺激CD28结扎，和细胞因子信号2个不同的谱系。 1型辅助细胞（Th1细胞）的特征在于它们的效应产生的IFNγ细胞因子以及它们的分化过程3,4中要求的IL-12信号传导。最终人们发现分化的Th1细胞具有遗传图谱是最鲜明特征BŸ的T盒家族转录因子的表达，TBX21（T-BET），这被认为是Th1细胞的遗传程序5的主调节器。此外，IL-12以及IFNγ可促进T-bet的表达6,7。在免疫应答，Th1细胞在针对细胞内病原体以及自身免疫性炎症的强启动子的宿主防御很重要。与此相反，2型辅助细胞（Th2细胞）要求的IL-4对它们的发展及其效应器细胞因子，包括IL-4，IL-5，和IL-13，是用于驱动B细胞反应的重要和致病在过敏8， 9。类似的Th1细胞，发现Th2细胞表达自己的主转录调节，称为GATA-3 10,11。有趣的是，偏振细胞因子的存在和特定的Th谱系的产生是对立对他人2,12的发展，表明只有一个特定的Th子集可以在免疫重新成为主导sponse。 由于Th1和Th2细胞谱系的鉴定，进一步的工作表明，甚至更独特的子集的辅助性T细胞，包括滤泡辅助（TFH），IL-9产生（TH9），和IL-22产生（TH22）（最近13检讨）。对于在体外分化实验的目的，该协议将只集中于两个附加Th亚群，被称为调节性T细胞（Treg）和IL-17的CD4 + T细胞（Th17细胞）。 CD25 +调节性T细胞可以在胸腺中自然发生（nTreg）;幼稚Th细胞也可以被诱导（iTreg）成为规中的周边（在14,15中综述）。这两种类型的调节性T细胞表达的特性的转录因子，称为叉头框P3（Foxp3的）这对于包括可溶性抗炎介体的生产，IL-2的消耗，以及细胞接触依赖性机制14,15其效应抑制机制的关键。 Foxp的缺乏在严重的，多器官的自身免疫紊乱3表达的结果称为免疫失调，多内分泌腺病，肠病，X连锁综合征（IPEX），这表明这钍子集在解决炎症和调节外周耐受自我的关键作用抗原16。在体外，天真的CD4 + T辅助细胞上调Foxp3的和刺激后成为致力于调节性T细胞的程序与IL-2和TGF-β14,15。有可能是适度的，以相当大的可塑性中的CD4 + T细胞谱系，只考虑细胞因子的产生（在17,18综述）时尤其如此。然而，对于在体外分化的协议的目的，我们将要讨论的每个子集作为一个独特的谱系。 最近，Th17细胞产生的IL-17的细胞因子的一个子集被确定为一个独特谱系与促炎性功能是特别致病过程中的自身免疫性炎症19-21。 Th17细胞表达一种独特的转录因子，被称为维甲酸相关孤儿受体γ吨（RORγt）的协调Th17细胞的遗传程序22。 TGFβ是Th17细胞谱系的产生重要通过RORγt的诱导。然而，TGFβ信号传导的效应被认为是只诱导时与IL-6（在12中综述）协同作用的Th17承诺。进一步的研究表明，各种其它信号，可以正调控的Th17承诺，包括IL-1β，增加的钠和TLR信号23-26。其它报告建议，致病Th17细胞在体内是那些实际上绕过TGFβ信号传导，而是依赖于IL-1，IL-6，和IL-23的其分化27的组合。因此，Th17细胞可衍生自各种信号通路;此协议的目的，常用（TGFβ和IL-6）途径Th17细胞谱系的承诺将提交​​。 下面描述的所有效应器谱系分化协议依靠固定抗体上进行刺激的T细胞受体和CD28整个实验的全过程。然而，其他人已经表明，TCR活化与抗原呈递细胞28或交联的抗CD3和与仓鼠抗体抗CD28抗体2天29顷也诱导各种Th亚群的产生的非常有效的手段。这里介绍的协议建立在用于从次级淋巴器官30分离鼠CD4 + T细胞，并产生Th17细胞31以前报道的方法。一个主要的区别是，此协议依赖于使用细胞分选仪的从淋巴组织分离幼稚CD4 + T细胞。然而，许多公司现在提供快速分离试剂盒，可以富集幼稚CD4 + T细胞，其可以是能够绕过要求˚F或排序取决于实验。的方法和在这个协议中提出的试剂是我们经常使用并且发现是最有效的。但是，请记住，替代试剂和方法很多下面介绍的步骤存在，它是由单独的实验室，以确定哪些工作最适合自己的目的。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Complete RPMI:</td>
			<td></td>
			<td></td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Life Technologies</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>10 % FBS</td>
			<td>Life Technologies</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>1000X 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>100X Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>100X L-glutamine</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>120 micron nylon mesh</td>
			<td>Amazon</td>
			<td>CMN-0120-10YD</td>
			<td>Cut into 2 cm2 squares and autoclave</td>
		</tr>
		<tr>
			<td>Alternative: 100 micron cell strainers</td>
			<td>Fisher</td>
			<td>08-771-19</td>
			<td>Alternative to cutting nylon mesh</td>
		</tr>
		<tr>
			<td>autoMACS running buffer</td>
			<td>Miltenyi</td>
			<td>130-091-221</td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>autoMACS rinsing solution</td>
			<td>Miltenyi</td>
			<td>130-091-222</td>
			<td>Warm in a 37 oC water bath before use</td>
		</tr>
		<tr>
			<td>CD4 beads</td>
			<td>Miltenyi</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysis buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokines:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human (h) IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse (rm) IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr>
			<td>rmIL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML-025</td>
			<td></td>
		</tr>
		<tr>
			<td>rmIL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr>
			<td>hTGFb</td>
			<td>R &#38; D Systems</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2C11 (anti-CD3)</td>
			<td>BioXcell</td>
			<td>BE0001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>37.51 (anti-CD28)</td>
			<td>BioXcell</td>
			<td>BE0015-1</td>
			<td></td>
		</tr>
		<tr>
			<td>11B11 (anti-IL-4)</td>
			<td>BioXcell</td>
			<td>BE0045</td>
			<td></td>
		</tr>
		<tr>
			<td>XMG1.2 (anti-IFNg)</td>
			<td>BioXcell</td>
			<td>BE0055</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD62L-FITC</td>
			<td>BioLegend</td>
			<td>104406</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>anti-CD25-PE</td>
			<td>BioLegend</td>
			<td>102008</td>
			<td>Use at 1:400</td>
		</tr>
		<tr>
			<td>anti-CD4-PerCP</td>
			<td>BioLegend</td>
			<td>100434</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>anti-CD44-APC</td>
			<td>BioLegend</td>
			<td>103012</td>
			<td>Use at 1:500</td>
		</tr>
		<tr>
			<td>Phorbol&#160; 12-myristate 13 acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>P-8139</td>
			<td>Prepare a stock at 0.1 mg/ml in DMSO and freeze aliquots at -20 oC</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I-0634</td>
			<td>Prepare a stock at 0.5 mg/ml in DMSO and freeze aliquots at -20 oC</td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>eBioscience</td>
			<td>00-4506-51</td>
			<td>Use at 1:1000</td>
		</tr>
	</tbody>
</table>

mouse na&#239;ve cd4+ t cell isolation and in vitro differentiation into t cell subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

分化可以根据条件的Th变化的分析的时间点被测试，以及T细胞受体激活的强度。后分化2-3天，细胞可以通过光学显微镜进行可视化，以确定的T细胞增殖的程度。井呈现细胞的广泛的增殖和结块很可能将是准备用于分析在4天分化的条件依赖于外源性的IL-2，如Th1和Th2，将后在培养4天后可能用尽介质。为了最大限度地提高细胞因子产生，如井枯竭介质可被补充新的RPMI含有IL-2或计数并重新铺板以每孔1...

Watch this Scientific Journal Video about Mouse NaÃƒÂ¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets at JoVE.com

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

mouse-nave-cd4-t-cell-isolation-vitro-differentiation-into-t-cell

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Immunology and Infection

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

51.7K 次观看.  Rosalind Franklin University of Medicine and Science. Naïve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of naïve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals. 

视频: Mouse Naïve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

枯否细胞分离的纳米粒子毒性测试

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.