这项研究由 JDRF 1-2014-243-N, ADA 7-14-JF-07, NIH 5 P30 DK079638-05 飞行员 PJ, 和罗伯特和珍妮丝捷基金会资助。
我们感谢桑德拉和 Andrene 麦当劳的耐心招聘, 塞缪尔. 布鲁姆为技术援助, Dr. 乔治 Makedonas 的礼物抗体和控制 DNA 用于 PCR 优化。

1. 确定感兴趣的 T 细胞群体. 
 注意: 抗原特异增殖与细胞分裂染料 (如羧基琥珀酰亚胺酯, CFSE) 可用于分离 T 细胞的基础上, 其增殖反应抗原刺激。如果从 PBMCs 开始, 7 天的 体外 扩展应该足以识别抗原特异的 CFSE 低种群 12 。
<ol> <li> 将 PBMCs 1:1 与 MHC 匹配的进纸单元混合, 并使用5和 #181 的标签; M CFSE 细胞分裂染料. </li>
	<li> 板 2-2. 5 x 10 5 每个井在 96-井圆底培养板中的每个单元格200和 #181; L 10% FCS 完整的 RPMI 介质. 
	注: 完整的 RPMI 含有2毫米 l-谷氨酰胺, 1 毫米丙酮酸钠, 100 和 #181; M 不必要的氨基酸, 5 毫米 HEPES, 5.5 和 #215; 10 和 #8722; 5 单位 2-基, 100 U mL 和 #8722; 1 青霉素和100和 #181; g mL 和 #8722; 1 链霉素 </li>
	<li> 用 25 #181; M 肽抗原刺激细胞. </li>
	<li> 在第四天的文化, 小心删除和更换100和 #181; L 的媒体. 
	注: 抗原特异 T 细胞识别的一种更为优化的方法是使用肽 HLA 体 13 。聚丙烯染色允许同时评估抗原特异性以及 HLA 限制. </li>
</ol> 2。设置单个单元格排序. 
<ol> <li> 执行引擎盖或指定区域中的所有过程, 没有放大的模板。分的试剂, 以避免污染, 清洁所有的工作表面, 如果可行, 设备与10% 漂白剂之前, PCR 设置。使用过滤提示, 核糖核酸和 dnasei 免费试剂和塑料在所有的 PCR 和载体克隆步骤。关键试剂可以在 材料表 中找到. 
	注意: 多重 PCR 反应是高度敏感的, 低水平的污染会导致产品的放大. </li>
	<li> 通过将 10x rt 反应缓冲、25X DNTPs、9 u rt 酶、0.01% 海卫 x、0.7 u 核糖核酸抑制剂和 383 nM 的每个 rt TCR 底漆 (列于 表 1 ) 中, 在无菌移油藏中生成反向转录主混合。对于每96个井板, 组成足够的主混合10额外的反应 (共 106), 以弥补液体损失在移。请参见 表 2 , 以了解反应组件和每井的金额. </li>
	<li> 准备一个96孔 PCR 收集板的细胞分类由移6和 #181; L 的反向转录主混合每井与多通道吸管。把盘子放在冰上或放在寒冷的街区. </li>
	<li> 将带有抗体的细胞标记为细胞表面标志物 (如 CD4 和 CD3, 或 CD8 和 CD3, 分别在2和 #181; g/毫升, 结合细胞分裂染料或聚丙烯染色). </li>
	<li> 对每个单元格中的 T 单元格进行排序, 跳过第十二行, 这将作为一个负控制. 
	注意: 排序的效率将取决于在板持有人的精确收集板对齐, 以及保持在一个较低的温度下的细胞和板块在 cDNA 反应之前。收集板应保持在冰的时刻, 除了在排序, 当他们固定在冷却板。并非所有机都装有温度调节板架;但是, 强烈建议使用一种。由于该类通常需要约 10-15 分钟每盘, 有一个增加的潜力, RNA 退化, 如果该板块不保持在低温. </li>
	<li> 作为一个正向控制, 在这个阶段用吸管将大量的细胞 (100 细胞) 手动添加到其中的一个井中。另外, 以前分离的 RNA 从汇集的细胞可以使用作为正面控制在 10 ng 每井. 
	注: 为了避免其他井的交叉污染和假阳性结果, 移液管精确、小心地纯化了 RNA. </li>
	<li> 用胶膜封板, 在 1000 x g 处旋转5分钟, 在4和 #176; C. </li>
	<li> 将 RNA 转录到25和 #176; c 为 10 min、37和 #176; c 为 45 min、85和 #176; c 为 10 min. 
	注意: 为了保证有效的 pcr 反应, 建议在同一天进行第一次 pcr 反应。另外, 转录板可以保存在-80 和 #176; C 最多2月. </li>
</ol> 3。执行多重嵌套 PCR 注意: 准备所有的主混合在一个干净的无模板区, 以避免污染. 
<ol> <li> 将每个可变区域底漆重新重建为100和 #181; M 与 dnasei 和核糖核酸自由 H 2 O。接下来, 结合20和 #181; L 每个44引物生成 V 型α入门池。生成 v 型β-beta 入门池结合20和 #181; l 每 40 v 型β底漆加上80和 #181; l dnasei 和核糖核酸自由 H 2 o. 底漆序列在 表 1 中列出。一旦汇集, 每引物的集中在混合是2.3 和 #181; m. </li>
	<li> 将常量区域底漆重建为100和 #181; m 库存解决方案与 dnasei 和核糖核酸自由 H 2 o. 将常量区域底漆稀释到10和 #181; m 工作解决方案和分使用. </li>
	<li> 准备两个单独的主混合 TCR-α和 TCR-β放大。通过合并5X 缓冲区来生成主混合80和 #181; m dNTPs, 3% 亚砜, 2 和 #181; m 底漆池 (每个底漆的最终浓度 46 nm), 200 nm TCR-恒定区域反转底漆, 1 U DNA 聚合酶, dnasei 和核糖核酸自由 H 2 O ( 表2/强 >)。为每96个井板, 构成足够的主混合为10另外的反应 (共计 106), 为了补偿液体损失在移期间. 
	注: 计算依据的是25和 #181 的最终 pcr 量; 每个井, 22.5 和 #181; i. pcr 组合与2.5 和 #181; l cDNA 模板. </li>
	<li> 在冰上或在冷块中保留该板的 cDNA。使用多通道, 以吸管22.5 和 #181; L TCR β反应到一个新的96井 PCR 板。然后使用多通道传输2.5 和 #181; L cDNA 进入 PCR 板. </li>
	<li> 使用多通道吸管22.5 和 #181; L TCR α反应直接进入含有其余的 cDNA 的板块. </li>
	<li> 用粘合剂将板材密封, 轻轻涡旋, 然后在 1000 x g 处旋转5分钟, 在4和 #176; C. </li>
	<li> 运行多重 PCR 反应与以下周期: 5 分钟在95和 #176; c 和34周期二十年代在95和 #176; c, 三十年代在54和 #176; c, 1 min 在72和 #176; c, 7 min 在72和 #176; c. </li>
	<li> 在25和 #181 的最终容量中进行嵌套 pcr 反应; 使用2.5 和 #181; 采用多重 pcr 混合的 l 作为模板。
	<ol> <li> 将内部和适配器的底漆重新制作为100和 #181; m 与 dnasei 和核糖核酸自由 H 2 o. 10 和 #181; 等分为工作库存. </li>
		<li> 准备两个单独的主混合 TCR-α和 TCR-β ampl分类.混合5X 缓冲器, 25X DNTP, 3% 亚砜, 200 nm 前向适配器底漆, 200 nm 反向嵌套底漆, 1 U dna 聚合酶, 和 dna 聚合酶和无核酸的 H 2 O 为最后的容量22.5 和 #181; L ( 表 2 )。对于每96个井板, 组成足够的主混合10额外的反应 (共 106), 以弥补液体损失在移. 
		注: 在第二 pcr 反应中使用 5X pcr 缓冲含有负载染料, 以方便琼脂糖凝胶负载. </li>
		<li> 使用多通道, 吸管的主混合为 TCR α和 TCR β反应在22.5 和 #181; L 每井成两个新的 96-井 PCR 板. </li>
		<li> 在2.5 和 #181 添加多重 PCR 反应模板; 每井. 
		注: 其余第一个 PCR 反应应密封, 并存储在-20 和 #176; C 作为附加模板的源, 如果需要 (请参见 步骤4.3 注意 ). </li>
		<li> 密封板, 轻轻涡, 并在 1000 x g 旋转下来5分钟, 在4和 #176; C. </li>
		<li> 运行带有以下循环的嵌套 PCR 反应: 5 分钟在95和 #176; c 和34周期二十年代在95和 #176; c, 三十年代在56和 #176; c, 1 min 在72和 #176; c, 7 min 在72和 #176; c. </li>
		<li> 运行5和 #181; L 最后的反应, 在1% 琼脂糖凝胶含有溴溴 (包括负和阳性控制井). 
		注: 预计波段应运行约 500 bp 大小。 图 2 中显示了一个示例反应. </li>
		<li> 纯化第二反应的其余部分 (20 和 #181; l) 使用96井格式 PCR 纯化试剂盒, 洗与15和 #181; l 70 和 #176; C H 2 O 每井, 并执行桑格测序。使用适当的 TCR-α或 TCR-β适配器前向引物进行测序 ( 表 1 ). 
		注意: 可以使用在线免疫软件分析序列. </li>
	</ol> </li> </ol> 4。亚克隆α和β PCR 链. 
 注: 第一反应中使用的正向引物的池, 第二个 PCR 反应中的反向引物用于合并限制部位。因此, 所获得的α和β链 PCR 产品可以按顺序 sub-cloned 到模板反转录病毒载体 ( 图 1 ). 
<ol> <li> 使用 BstbI 和 MfeI ( 表 3 ) 从 beta 嵌套反应中摘录纯化的 PCR 产品. 
	注: 请勿超过 5 #181; g 插入. </li>
	<li> 并行, 用 BstbI 和 MfeI ( 表 3 ) 来消化模板向量。使用1-2 和 #181; g 的模板向量每 TCR。在琼脂糖凝胶上运行的消化载体, 并隔离较大的波段 (〜 7500 bp)。用凝胶 DNA 纯化试剂盒净化载体. 
	注: 在限制酶消化期间, 模板小鼠 TCR-β变区被删除, 允许增加人类 TCR-β变量区域. </li>
	<li> 使用 pcr 纯化试剂盒提纯经消化的 pcr 产物. 
	注: 验证套件中所含列的 DNA 纯化能力;必要时使用多个列。成功结扎反应所需的纯化插入物的最小用量为 50 ng, 最低浓度为 10 ng/和 #181;如果纯化的插入量是不足够的成功结扎, 第二个 PCR 反应可以重复. </li>
	<li> 将模板向量与 10 U CIP 酶用于1小时的37和 #176; c 以防止 self-ligation, 其次是热失活10分钟, 在75和 #176; c. 用 PCR 纯化试剂盒提纯 DNA ( 表 3 ). </li>
	<li> 结扎 TCR-β插入和向量, 使用 DNA 连接在20和 #181; L 总体积在6摩尔, 超过30分钟的插入物在室温下. 
	注: 总的说, 结扎反应应包含约 150 ng 的 DNA ( 表 3 ). </li>
	<li> 用于转换, 混合8和 #181; 80 和 #181 的结扎反应 l; 具有化学能力的 大肠杆菌 DH5 和 #945; 细胞 (材料的 表 ) 和在冰上孵育30分钟的热休克在42和 #176; C 为四十五年代. 添加500和 #181; 我超级物抑制 (soc) 培养基的最佳肉汤, 在37和 #176 时摇动1.5 小时; C. </li>
	<li> 将250和 #181; 在含有性汤 (LB) 琼脂板的氨苄西林中培养细菌。在37和 #176 的夜间 (约16小时) 孵育盘; C. </li>
	<li> 第二天选择细菌菌落, 并在一夜之间在含有氨苄西林的3毫升培养基中生长。用 dna 质粒纯化试剂盒分离质粒 dna. </li>
	<li> 确认通过 small-scale 测试-摘要成功插入 beta 链, 并使用限制酶 BstbI 和 MfeI。在12和 #181; l 总反应量, 结合200-500 和 #181; g 质粒 DNA, 0.25 和 #181; 每种酶的 l, 和10X 酶缓冲 ( 表 3 )。运行的反应1% 琼脂糖凝胶含有溴溴, 以确认大小的插入带 (〜 500 bp). </li>
	<li> 在确认插入的存在后, 使用 IRES 反向底漆 ( 表 1 ) 序列化向量的 TCR-beta 部分. </li>
	<li> TCR-beta 插入的顺序确认后, 执行类似的过程以插入 alpha 变量区域。从α嵌套反应中提炼纯化的 PCR 产物, 以及新生成的 TCR-β插入载体, 其中含有 SnaBI 和 SacII. 
	注: 在限制酶消化期间, 模板小鼠 TCR α变区被移除, 允许增加人类的 TCR-α变量区域. </li>
	<li> 结扎在20和 #181 中使用 DNA 连接对相应的 beta 变量区域进行编码, 并将其插入到 TCR 向量中; 在室温下, 6 摩尔的总体积超过30分钟的插入量. 
	注: 总的说, 结扎反应应包含约 150 ng 的 DNA ( 表 3 ). </li>
	<li> 用于转换, 混合8和 #181; 80 和 #181 的结扎反应 l; 具有化学能力的 大肠杆菌 DH5 和 #945; 细胞 (材料的 表 ) 和在冰上孵育30分钟的热休克在42和 #176; C 四十五年代. 添加500和 #181; 我 soc在37和 #176 时为1.5 小时的介质和抖动; C. </li>
	<li> 将250和 #181; 在含有性汤 (LB) 琼脂板的氨苄西林中培养细菌。在37和 #176 的夜间 (约16小时) 孵育盘; C. </li>
	<li> 第二天选择细菌菌落, 并在一夜之间在含有氨苄西林的3毫升培养基中生长。用 dna 质粒纯化试剂盒分离质粒 dna. </li>
	<li> 确认将α链成功插入的 small-scale 测试摘要与限制酶 SnaBI 和 SacII。在12和 #181; l 总反应容量结合200-500 和 #181; g 质粒 DNA, 0.25 和 #181; 每种酶的 l, 和10x 酶缓冲 ( 表 3 )。运行的反应1% 琼脂糖凝胶含有溴溴, 以确认大小的插入带 (〜 500 bp). </li>
	<li> 在确认插入的存在后, 使用 MSCV2-forward 入门 ( 表 1 ) 将向量的 TCR-alpha 部分序列化. </li>
	<li> 一旦验证了 alpha 链序列, 请通过 TCR-β-反转和 IRES 反向底漆 ( 表 1 ) 的顺序验证完整的 TCR 插入. </li>
</ol> 5。验证 TCR 细胞表面的表达和特异性. 
 注意: HEK293T 单元用于测试成功的 TCR 链ng 和单元格表面表达式 ( 图 3A ) 8 。多肽 MHC 聚丙烯染色也可以对转染的 HEK293T 细胞进行测试的保留抗原特异性后 TCR 基因分离 ( 图 3B ). 
<ol> <li> 将12个组织培养皿中的 HEK293T 细胞在1毫升的 1 x 10 5 细胞中, 在5% 和 #176 的完整 DMEM 介质中包含37个 FCS 和区域性; c. 板出足够的细胞为每个新的 TCR 指定单独的井, 控制模板 TCR 矢量, 仅 CD3 向量, 控制 TCR 向量, non-transfected. 
	注: 用完整的鼠标模板 TCR 载体转染 (纶) 与 mCD3 和 #949 结合; #947; #948; #950; pMIG 向量, 单纶向量, mCD3 #949; #947; #948; #950;-pMIG 矢量单独被用作正负控制. 
	注: 完整的 DMEM 包含2毫米 l-谷氨酰胺, 1 毫米丙酮酸钠, 100 和 #181; M 非必需氨基酸, 5 毫米 HEPES 缓冲, 5.5 x 10 -5 单位 2-基, 100 U/毫升青霉素/链霉素. </li>
	<li> 第二天, 染1和 #181 的单元格; 纶向量、1和 #181; g 鼠标 CD3 和 #949; #947; #948; #950;-pMSCVII-GFP (pMIG) 载体, 和 liposome-based 转染试剂按照制造商的指示. </li>
	<li> 对于每个 TCR, 添加6和 #181; 将试剂转染为100和 #181; l 室温, 无血清 DMEM。短暂的旋涡, 并在室温下孵育15分钟. </li>
	<li> 在单独的1.5 毫升管中结合1和 #181; g TCR 纶向量与1和 #181; g 鼠标 CD3 和 #949; #947; #948; #950;-pMSCVII-GFP (pMIG) 矢量, 或1和 #181; 每个矢量的 g 分别用于控件. </li>
	<li> 滴, 将转染试剂/DMEM 溶液添加到包含矢量 DNA 的管中。室温孵育15分钟. </li>
	<li> 滴, 将转染试剂/DMEM/DNA 溶液添加到 HEK293T 细胞中。轻轻地轻敲盘子混合。孵育在37和 #176; C 为 24 h. </li>
	<li> 在24小时后替换媒体, 并将单元格保留为24小时. </li>
	<li> 通过大力移将细胞从板上移除, 并用 anti-mouse CD3 抗体 (2.5 和 #181; g/毫升) 对细胞进行染色, 以验证流式细胞术对嵌合 TCR 的表面表达. </li>
	<li> 为了比较被转染为类似水平的单元格之间的 CD3 表达式, 对表达类似高水平荧光报告分子的细胞进行分析 (GFP 为 CD3 复合体, 石为 TCR 表达载体)。优选地, 分析应该在 10 4 到 10 5 荧光强度 ( 图 3A ) 内的 GFP + 石 + 细胞上进行封闭. 
	注: 在转嵌的细胞中的 CD3 表达水平应与控制鼠标 (原始模板) 矢量 ( 图 3A ) 相媲美。生成的 TCR 反转录病毒载体与先前描述的 4 中的 在体内 表达式系统兼容。
</ol>

<ol>
	<li>Germain, R. N. <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+development+and+the+CD4-CD8+lineage+decision.">T-cell development and the CD4-CD8 lineage decision.</a> Nat Rev Immunol. 2 (5), 309-322 (2002).</li><li>Singer, A., Adoro, S., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+fate+and+intense+debate:+myths,+models+and+mechanisms+of+CD4-+versus+CD8-lineage+choice.">Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.</a> Nat Rev Immunol. 8 (10), 788-801 (2008).</li><li>Tubo, N. 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=Single+naive+CD4++T+cells+from+a+diverse+repertoire+produce+different+effector+cell+types+during+infection.">Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.</a> Cell. 153 (4), 785-796 (2013).</li><li>Sprouse, M. 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=Rapid+identification+and+expression+of+human+TCRs+in+retrogenic+mice.">Rapid identification and expression of human TCRs in retrogenic mice.</a> J Immunol Methods. , (2016).</li><li>Lennon, G. 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=T+cell+islet+accumulation+in+type+1+diabetes+is+a+tightly+regulated,+cell-autonomous+event.">T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.</a> Immunity. 31 (4), 643-653 (2009).</li><li>Dash, 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=Paired+analysis+of+TCRalpha+and+TCRbeta+chains+at+the+single-cell+level+in+mice.">Paired analysis of TCRalpha and TCRbeta chains at the single-cell level in mice.</a> J Clin Invest. 121 (1), 288-295 (2011).</li><li>Kobayashi, 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=A+new+cloning+and+expression+system+yields+and+validates+TCRs+from+blood+lymphocytes+of+patients+with+cancer+within+10+days.">A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.</a> Nat Med. 19 (11), 1542-1546 (2013).</li><li>Szymczak, A. 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=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat Biotechnol. 22 (5), 589-594 (2004).</li><li>Szymczak, A. L., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Bettini, M. L., Bettini, M., Vignali, D. A. <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+receptor+retrogenic+mice:+a+rapid,+flexible+alternative+to+T-cell+receptor+transgenic+mice.">T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3 (3), 191-197 (2006).</li><li>Mannering, S. 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=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J Immunol Methods. 283 (1-2), 173-183 (2003).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Corthay, A., Nandakumar, K. S., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+percentage+of+peripheral+T+cells+with+two+different+T+cell+receptor+alpha-chains+and+of+their+potential+role+in+autoimmunity.">Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.</a> J Autoimmun. 16 (4), 423-429 (2001).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <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+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> J Vis Exp. (113), (2016).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <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+T+cell+receptor-retrogenic+mice:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8 (10), 1837-1840 (2013).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66 (17), 8878-8886 (2006).</li></ol>

作者没有什么可透露的。

在当前的协议中, 我们描述了一个有效的方法单细胞 TCR 放大和随后的 sub-cloning 配对 TCR α和β链成模板逆转录病毒表达载体。虽然, 已经开发了几个单细胞 PCR 协议, 到目前为止没有与直接 sub-cloning 兼容到表达载体。在大多数情况下, 包含高度可变的 CDR3 区域的部分序列被放大, 具有足够的序列来推断变量区域。这种较小的 pcr 产品尺寸支持更高的 pcr 效率, 但需要de 从头TCR 基因合成功能研究?...

t 细胞受体 (TCR) 指示 t 细胞命运决定在发展期间, 稳态/稳态, 和抗原刺激的外围1,2,3。最近的深度测序技术的扩展揭示了先前未被低估的抗原特异 T 细胞反应中的 TCR 多样性。TCR 多样性表明了功能广泛的 T 细胞反应的潜力。为了将 TCR 序列剧目分析与 TCR 功能分析相结合, 测序方法应设计为与实验系统兼容, 并用于后续的功能性研究的体内模型。TCRs我们已经开发了一个有效的方法, 人类 TCR 序列隔离和简化 sub-cloning 成嵌合体的人/小鼠 TCR 模板载体与 TCR 表达的 HLA-人性化的老鼠4。heterodimeric TCRs 的相应α和β链的分离需要从单个细胞中扩增两个链。虽然, 已经开发和利用了几个单细胞的 TCR 克隆协议, 但迄今为止, 没有一个能够很容易地与高通量简化的 Vα/Vβ TCRs 的直接克隆相兼容, 成为重新体内的反转录病毒载体 4、5、6、7。以前的研究采用了两种主要方法, 要么是有选择地放大 TCR 的有限部分足以推断序列, 要么放大整个 TCR 序列4,5,6,7. 通过第一种方法获得的 TCR 序列的下游功能分析需要昂贵的在硅片中程序集和de 从头构造 TCR。虽然第二种方法提供完整的 TCR 序列, 但人的恒定区域与在鼠标模型中克隆的 TCRs 的在体内表达不兼容。我们的方法是专门设计为与在体内的功能分析 TCRs 在小鼠模型中兼容。我们开发了一个高效和简化的单细胞 pcr 协议, 允许直接 sub-cloning pcr 片段到模板表达载体。
我们的方法采用了一个高度敏感的多重嵌套 PCR 反应, 这是两个步骤执行。在第一个复用反应步骤中, 所有 v-β链的40引物的池, 以及所有 v 型α链专用的44个引物池, 用于放大 TCR-α或 TCR-beta, 而无需事先了解序列 (表 1)。前引物有一个适配器序列, 这是纳入 PCR 产品的 5 "。反向底漆基于 TCR 的恒定区域。我们已经确定了在人类变量或连接 TCR 区域中不存在的唯一限制站点, 并将它们合并到新重新设计的 TCRα和 TCRβ引物 (图 1) 中。在第二个嵌套反应中, 使用在恒定区域内的适配器序列和嵌套反向底漆的底漆, 用于进一步放大 TCR 链, 增加特异性 (表 1,图 1)。单细胞分离后, 两轮 pcr (第一反应与池的 Vα和 Vβ引物, 第二次与适配器引物) 产生的 pcr 产品, 可以直接 sub-cloned 到模板逆转录病毒载体。最后的 TCR 结构将编码, 在一个单一的开放阅读框架, 人的可变区域结合的小鼠恒定区域连接的 ' self-cleaving ' 蛋白序列, P2A (hVα-mCα-P2A-hVβ-mCβ)8。P2A 序列已在多个系统中使用, 并已被广泛测试, 专门用于表达 TCRs8、9、10、11。虽然, 在翻译以后多数 P2A 序列仍然附有由一个灵活的链接器连接的阿尔法链子的 C 末端, 而 beta 链信号序列有另外的脯氨酸, 这种修改对 TCR 作用没有有害作用。在构造中使用鼠标常量区域, 而不是人类, 以避免 re-expressed 在鼠标单元格中时与下游信号组件的潜在改变的交互。单一的, 将导致化学计量独立表达α和β链9,11。目前的协议是基于广泛可用的试剂和方法, 并且设计为以流线型和高效的方式进行。虽然我们已经明确地使用这项技术来检测 TCRs 从反应 T 细胞牵连自身免疫性糖尿病, 我们预计, 该协议可以广泛适用于识别和功能评估的人 TCRs 具体自身免疫表位, 肿瘤抗原表位, 或对病原体和疫苗的反应。

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CFSE&#160;</td>
			<td style="width:148px;">eBioscience&#160;</td>
			<td style="width:116px;">65-0850-84</td>
			<td style="width:223px;">5 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD4 (OKT4) BV421</td>
			<td>Biolegend&#160;</td>
			<td>317433</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD3 (OKT3) PerCP/Cy5.5</td>
			<td>Biolegend&#160;</td>
			<td>317336</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD3 AF647</td>
			<td>Biolegend&#160;</td>
			<td>100322</td>
			<td>2.5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant RNAse inhibitor biotech grd 2.5KU&#160;&#160;</td>
			<td>VWR</td>
			<td>97068-158</td>
			<td>0.7 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Applied Biosystems High capaity cDNA RT kit&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>4368814</td>
			<td>9 U (RT enzyme)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NF-H2O: Nuclease-free water 1000mL</td>
			<td>Invirtogen&#160;</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gotaq DNA polymerase 2500U</td>
			<td>VWR</td>
			<td>PAM3008</td>
			<td>1 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Half Skirt PCR Plate</td>
			<td>Phenix</td>
			<td>MPX-96M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp Clear Adhesive Film&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>4306311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SnaBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0130</td>
			<td>15 U (insert) 30 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacII</td>
			<td>New England Biolabs&#160;</td>
			<td>R0157</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MfeI</td>
			<td>New England Biolabs&#160;</td>
			<td>R3589S</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BstBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0519</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calf Intestinal Phosphatase (CIP)</td>
			<td>New England Biolabs&#160;</td>
			<td>M0290S</td>
			<td>10 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quick ligase&#160;</td>
			<td>New England Biolabs&#160;</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus minipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A1460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus midipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A7640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA clean &#38; concentrator-25 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-304C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZR-96 DNA clean &#38; concentrator-5 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-306A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subcloning Efficiency DH5a Competent Cells&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transit LT-1 Reagent 1mL (transfection reagent)</td>
			<td>Mirus&#160;</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Aria II (sorter)</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Fortessa (flow cytometer)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs

虽然, 有几种方法的排序的配对 t 细胞受体 (TCR) α和β链从单一 t 细胞已经开发, 迄今没有有利于下游体内功能分析的 TCR heterodimers。我们已经开发了一个改进的协议的基础上 two-step 多重嵌套 pcr, 这导致一个 pcr 产品横跨整个可变区域的人类 TCR α和β链。通过确定独特的限制点, 并将它们纳入 pcr 引物, 我们已经使 pcr 产品与直接 sub-cloning 与模板逆转录病毒载体相兼容。由此产生的逆转录病毒构建编码一个嵌合的人/鼠 TCR 与鼠标的细胞内域, 这是功能在鼠标细胞或在体内鼠标模型。总的来说, 这里描述的协议结合了人类单细胞配对 TCR α和β链识别与简化的逆转录病毒载体易于适应体外和体内TCR 表达。视频和伴随的材料被设计为对单细胞 PCR 的高度详细的描述, 因此关键的步可以被跟随和潜在的陷阱避免。此外, 我们还提供了生成表达载体所需的克隆步骤的详细描述。一旦掌握, 从单细胞排序到 TCR 表达的整个过程可以在短短的两周内完成。

在步骤 3.2.7 (图 2) 中, 通过在琼脂糖凝胶上5µL 第二反应的运行, 对多重嵌套 PCR 反应的效率进行了检查。平均 TCR-β放大率的效率预计在80% 左右, 而 TCR-α反应的效率通常较低, 约 50%4。只有配对的 TCR α和 TCR β链可以用于 TCR 表达;然而, 所有 PCR 产品都可以测序, 以获得 TCR 序列和剧目信息。
<p class="jove_content" fo:keep-together.within-page="1"...

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Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

当前的协议结合了单细胞配对人类 TCR α和β链测序与与体外和体内TCR 表达相兼容的反转录病毒载体的生成。

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

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Immunology and Infection

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

9.3K 次观看.  Baylor College of Medicine, Texas Children's Hospital. 当前的协议结合了单细胞配对人类 TCR α和β链测序与与体外和体内TCR 表达相兼容的反转录病毒载体的生成。

视频: Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

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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 ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.