这项工作得到了国家卫生研究院 (T32 DA007097、R01AI096966、andUM1AI26617) 的公共卫生服务补助金的支持。

1. 1 天: 新鲜的组织切片和主要孵化 <ol> <li> 使用手术刀将新鲜的组织切成小 (大约0.5 厘米宽0.5 厘米高) 的碎片。分别将每个组织粘附到一个柱塞上, 并在 PBS 中嵌入 3-5 毫升的 4% low-melt 琼脂糖。用贴纸标记柱塞与组织信息。把它放在冰桶的冷冻柜里, 以固化. </li>
	<li> 打开切片并将剖面的粗细设置为200和 #181; m. 在切片上安装刀片式服务器, 并在切片浴缸中插入带有组织的柱塞. </li>
	<li> 用肝素 (pbs-H) 制备磷酸盐缓冲盐水, 加入100和 #181; 在 pbs 中添加 g/毫升或 18.7 U/毫升肝素以保存 RNA, 并允许下游潜在的应用。用 100-120 毫升的冷冻、无菌的 PBS 填充切片浴, 覆盖嵌入的组织。在浴缸中加入 PBS-H 冰块, 以保持 0-2 和 #176 的温度; c. 开始切片并将组织切成200和 #181; m 部分. 
	注意: 重要的是保持在冰上的组织冷冻, 以减少细胞活动的组织内, 因为新鲜的组织更容易切片时, 它是冷。如果没有针对下游的计划, 则可以单独使用 PBS. </li>
	<li> 或者, 对于不切片的组织 (例如, 肠道和肺部), 使用手术刀或刀片将组织切成薄条, 尽可能接近200和 #181; m. </li>
	<li> 用实验样本信息对24个组织培养皿的盖子进行标记, 并将组织室放置在相应的井中。使用画笔将剖面转移到一个24井组织培养板的井中, 其中含有1毫升的冷却 PBS-H. 
	注: 在开始染色前, 应先制作可重复使用的组织室。组织室可以使用14毫升聚丙烯圆底管和金属丝网。使用锋利的剃刀刀片削减底部的一个14毫升聚丙烯圆底的 snap 盖帽管。将金属丝网切割成一个圆, 以适应管子底部的孔。用本生燃烧器加热金属丝网圈, 直到 red-hot。将金属丝网圈迅速放下, 把管子推到网格上。检查金属丝网是否牢固地连接到管子的底部, 然后小心地用锋利的剃刀刀片在3毫升标记上切断管子的顶端。在每个组织室中放置多达3组织切片, 或每井组织多达 1 cm 2 。保持至少一个空井之间的不同的抗体组合, 以避免交叉污染. </li>
	<li> 在完成将所有切口部分转移到组织室后立即进行主聚丙烯和抗体染色。在任何时候保持1毫升的 PBS-H 的部分被淹没和冷冻. </li>
	<li> 在夜间用0.5 和 #181 孵育组织切片; FITC 共轭的、多肽负载的 MHC I 体在 PBS-H 中稀释, 2% 正常山羊血清 (NGS)。包括老鼠或其他 non-rabbit 抗体定向的细胞外表位的兴趣在这个孵化 ( 例如, 大鼠 anti-CD8 抗体稀释1:500 在 PBS H 与 2% NGS)。每井放置1毫升稀释的抗体. 
	注: 在选择 CD8 抗体时应注意, 一些可以增强, 有些可以抑制 MHC 体对 T 细胞受体的束缚 4 , 21 。在这里描述的大鼠人 CD8 抗体是不稳定的, 有时会导致一些微弱的染色。这是用于三重标记, 因为它是唯一的 non-rabbit 和 non-mouse CD8 抗体测试, 染色恒河猴 CD8 T 细胞. </li>
	<li> 使用1毫升的溶液每井的主要抗体和所有后续孵化, 并执行这一和所有随后的孵化在4和 #176; C, 在一个摇摆平台上的板块. 
	注: 组织应在会议厅自由浮动. </li>
</ol> 2。2天: 固定和二次孵化 <ol> <li> 在主孵化后, 用1毫升的冷冻 PBS 清洗两次, 每次洗涤20分钟。通过将组织腔转移到不同的24孔组织培养基中, 在相应的井中包含1毫升的冷冻 PBS H. 
	注意: 在组织室间移动时, 要注意不要将内容从一个实验样品滴入另一种。对于所有随后的孵化和洗涤, 类似的转移组织室到一个干净的板包含适当的解决方案。在手术过程中, 一定要对组织室的各部分进行监测, 以确保这些部分不会粘在组织室的两侧。如果他们这样做, 把他们推回解决方案. </li>
	<li> 用1毫升新鲜 PBS-缓冲4% 甲醛在室温下2小时修复部分 (不要 over-fix)。用冷的 PBS 冲洗两次, 5 分钟 
	警告: 甲醛有毒;穿戴适当的个人防护设备. 
	注: 如果需要抗原提取和性检测细胞表位, 抗原可以通过煮沸的切片0.01 摩尔尿素后, 甲醛固定. </li>
	<li> 将剖面转换为含有0.01 摩尔尿素的24井培养皿, 并将这些板放在微波炉中。煮沸的部分三次, 约十年代每个, 总共三十年代. 
	注意: 要非常小心, 煮沸的解决方案可以迫使部分出井。如果发生这种情况, 使用画笔将部分从组织室的两侧或从板的盖子推回到适当的组织室的底部. </li>
	<li> 在二次抗体孵化之前, permeabilize 并阻断组织切片, 将其孵化为含有 pbs-h、0.3% 洗涤剂 (pbs-T) 的阻断溶液和 2% NGS 在4和 #176 上的 1 H 的摇杆上; c. 执行随后的抗体孵化PBS-H-T/2%ngs. </li>
	<li> 对于次生孵育, 将组织室中的部分转移到含有家兔抗 FITC 抗体稀释1:10,000 的 PBS-H-T/2%ngs。夜间孵化. </li>
	<li> 在 PBS-H-T/2%ngs 中使用小鼠 anti-CD20 抗体稀释1:200 进行 counterstaining。对于这个选项, 如果需要的话, 检索表位, 渗透到细胞, 并在这种孵化之前阻止, 如上所述. </li>
</ol> 3。3天: 第三次孵化 <ol> <li> 在第二个孵育后, 在 PBS H 中以4和 #176 的三次清洗剖面; C 至少 20 min. </li>
	<li> 执行最后的孵化与适当的荧光标记抗体 ( 例如, 山羊兔共轭绿黄色, 山羊鼠共轭远红色染料, 和山羊 anti-mouse 共轭绿色染料抗体稀释 1:5, 000,1:5, 000, 和 1:2, 000, 分别在 PBS-H-T/2%ngs)。夜间孵化. 
	注意: 在这一点上, 孵化可以延长多达三天, 如果需要。在这个 incu 中, 通过在锡箔纸上包裹盘子来保持部分免受光线的保护。bation 步和尔后, 作为轻解渴荧光. </li>
</ol> 4。4天: 安装节 <ol> <li> 在 PBS H 中将部分三次洗涤, 至少 20 min. 
	注意: 如果计划在下游的 19 , 修复4% 甲醛中的部分, 1 小时, 以确保体和抗体到位, 然后清洗两次与 PBS h 的每节5分钟. 
	警告: 甲醛有毒;穿戴适当的个人防护设备. </li>
	<li> 使用画笔将剖面转移到显微镜幻灯片。注意不要把纸巾戳得太多。每节涂上甘油/明胶, 含有4毫克/毫升的无食子酸丙酯或另一种载有荧光防腐剂的安装介质。盖上片. </li>
	<li> 将幻灯片存储在-20 和 #176 的 light-protected 幻灯片容器中; c. 冲洗组织培养皿, 用酒精除去盖子上的标签. 
	注: 板可重复使用. </li>
</ol> 5。共焦显微镜图像的获取 <ol> <li> 使用共聚焦显微镜捕获高分辨率图像, 对每个荧光 ( 图 1A 和 B ) 采用适当的激光器和滤镜. 
	注: 在本例中, 使用了共焦显微镜 (参见 材料表 )。图像收集使用 561 nm 激光在20% 功率为绿色的黄色标记特异性 t 细胞, 488 nm 激光在10% 功率为绿标记的 CD20-expressing B 细胞, 和 640 nm 激光器在15% 功率为远红色标签的 CD8 t 细胞。20X 目标和数字孔径0.8 被使用了. </li>
	<li> 在多个 800 x 800 像素字段的三通道中依次收集 z 系列3和 #181; m (或其他) 间隔。构造收集字段的蒙太奇 ( 图 1C -e )。根据相应幻灯片的信息命名每个蒙太奇图像, 并保存以进行分析. </li>
	<li> 使用各自的共聚焦显微镜分析和量化软件或使用 ImageJ 进行量化图像分析. </li>
</ol> 6。定量图像分析 注意: 可以使用共聚焦显微镜分析和量化软件或使用 ImageJ 软件来完成定量图像分析。在这里, ImageJ 被用作一个例子. 
<ol> <li> 通过将其拖动到 ImageJ 窗口 ( 图 2A ) 来打开共焦蒙太奇. 
	注: ImageJ 可以直接打开蒙太奇收集的许多不同的共焦显微镜。如果 ImageJ 无法直接打开蒙太奇, 则将选定的 z 扫描作为 TIFF 文件导出以将其打开. </li>
	<li> 复制所选的 z 扫描以进行分析 (#34; 图像和 #34;-#62; #34;D 重复和 #34;) ( 图 2B ). </li>
	<li> 拆分不同的通道 (#34; 图像和 #34;-和 #62; #34; 颜色和 #34;-和 #62; #34; 分割通道和 #34;) ( 图 2C ). </li>
	<li> 在相应的通道中绘制量化分析的 roi 以实现目标, 并通过按下和 #34 将其添加到 roi 管理器中; #34; 在键盘上。测量该区域. 
	注: ImageJ 的 ROI 管理器显示了 #181 中的区域; m 2 ( 图 2D ). </li>
	<li> 调整要分析的通道的荧光亮度和对比度 (#34; 图像和 #34;-#62; #34; 调整和 #34;-#62; #34; 亮度/对比度和 #34;) ( 图 2E ). </li>
	<li> 拼合图像上的 roi (#34; roi 管理器和 #34; #62; #34; 拼合和 #34;) ( 图 2F ). </li>
	<li> 使用 and #34 量化图像中的正单元格; 多点和 #34; 工具 ( 图 2G ). </li>
</ol>

<ol>
	<li>Altman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li><li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+identify+peptide-specific+human+CD4(+)+T+cells+proliferating+in+response+to+influenza+A+antigen.">MHC Class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen.</a> J Clin Invest. 104, R63-R67 (1999).</li><li>Steinert, E. 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=Quantifying+memory+CD8+T+cells+reveals+regionalization+of+immunosurveillance.">Quantifying memory CD8 T cells reveals regionalization of immunosurveillance.</a> Cell. 161 (4), 737-749 (2015).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> J Immunol. 165 (2), 613-617 (2000).</li><li>Haanen, J. B., 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=In+situ+detection+of+virus-+and+tumor-specific+T-cell+immunity.">In situ detection of virus- and tumor-specific T-cell immunity.</a> Nat Med. 6 (9), 1056-1060 (2000).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tetramer+staining+using+MHC+class+I+tetramers.">In situ tetramer staining using MHC class I tetramers.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. 17, 4.1-4.9 (2004).</li><li>Li, 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=Use+of+HLA-DR*08032/E7+and+HLA-DR*0818/E7+tetramers+in+tracking+of+epitope-specific+CD4++T+cells+in+active+and+convalescent+tuberculosis+patients+compared+with+control+donors.">Use of HLA-DR*08032/E7 and HLA-DR*0818/E7 tetramers in tracking of epitope-specific CD4+ T cells in active and convalescent tuberculosis patients compared with control donors.</a> Immunobiology. 216, 947-960 (2011).</li><li>Bischof, 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=Analysis+of+autoreactive+CD4+T+cells+in+experimental+autoimmune+encephalomyelitis+after+primary+and+secondary+challenge+using+MHC+class+II+tetramers.">Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers.</a> J Immunol. 172, 2878-2884 (2004).</li><li>Massilamany, 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=Direct+staining+with+major+histocompatibility+complex+class+II+dextramers+permits+detection+of+antigen-specific,+autoreactive+CD4+T+cells+in+situ.">Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ.</a> PLoS ONE. 9, e87519 (2014).</li><li>Massilamany, 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=In+situ+detection+of+autoreactive+CD4+T+cells+in+brain+and+heart+using+major+histocompatibility+complex+class+II+dextramers.">In situ detection of autoreactive CD4 T cells in brain and heart using major histocompatibility complex class II dextramers.</a> J Vis Exp. (90), e51679 (2014).</li><li>Dileepan, T., Kim, H. O., Cleary, P. P., Skinner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Peptide-MHC-II+Tetramer+Staining+of+Antigen-Specific+CD4++T+Cells+in+Tissues.">In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.</a> PLoS ONE. 10 (6), e0128862 (2015).</li><li>Tjernlund, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+Gag-specific+CD8++cells+in+the+GI+tract+of+SIV+infected+Rhesus+macaques.">In situ detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques.</a> Retrovirology. 7, 7-12 (2010).</li><li>Connick, 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=CTL+fail+to+accumulate+at+sites+of+HIV-1+replication+in+lymphoid+tissue.">CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue.</a> J Immunol. 178, 6975-6983 (2007).</li><li>Hong, J. 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=Localized+Populations+of+CD8low/&#8722;+MHC+Class+I+Tetramer++SIV-Specific+T+Cells+in+Lymphoid+Follicles+and+Genital+Epithelium.">Localized Populations of CD8low/&#8722; MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium.</a> PLoS ONE. 4 (1), e4131 (2009).</li><li>Sasikala-Appukuttan, A. 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=Location+and+dynamics+of+the+immunodominant+CD8+T+cell+response+to+SIV&#916;nef+immunization+and+SIVmac251+vaginal+challenge.">Location and dynamics of the immunodominant CD8 T cell response to SIV&#916;nef immunization and SIVmac251 vaginal challenge.</a> PLoS ONE. 8 (12), e81623 (2013).</li><li>Connick, 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=Compartmentalization+of+simian+immunodeficiency+virus+replication+within+secondary+lymphoid+tissues+of+rhesus+macaques+is+linked+to+disease+stage+and+inversely+related+to+localization+of+virus-specific+CTL.">Compartmentalization of simian immunodeficiency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-specific CTL.</a> J Immunol. 193, 5613-5625 (2014).</li><li>Li, Q., 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=Visualizing+antigen-specific+and+infected+cells+in+situ+predicts+outcomes+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection.</a> Science. 323, 1726-1729 (2009).</li><li>Li, Q., Skinner, P. J., Duan, L., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+simultaneously+visualize+virus-specific+CD8++T+cells+and+virus-infected+cells+in+situ.">A technique to simultaneously visualize virus-specific CD8+ T cells and virus-infected cells in situ.</a> J Vis Exp. (30), e1561 (2009).</li><li>Li, 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=Simian+immunodeficiency+virus-producing+cells+in+follicles+are+partially+suppressed+by+CD8++cells+in+vivo.">Simian immunodeficiency virus-producing cells in follicles are partially suppressed by CD8+ cells in vivo.</a> J Virol. 90, 11168-11180 (2016).</li><li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide/major+histocompatibility+complex+multimers.">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> J Exp Med. 191, 335-346 (2000).</li><li>Lee, Y. J., Wang, H., Starrett, G. J., Phuong, V., Jameson, S. C., Hogquist, 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=Tissue+specific+distribution+of+iNKT+cells+impacts+their+cytokine+response.">Tissue specific distribution of iNKT cells impacts their cytokine response.</a> Immunity. 43 (3), 566-578 (2015).</li><li>Abdelaal, H. M., Kim, H. O., Wagstaff, R., Sawahata, R., Southern, P. J., Skinner, P. J. <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+Vibratome+and+Compresstome+sectioning+of+fresh+primate+lymphoid+and+genital+tissues+for+in+situ+MHC-tetramer+and+immunofluorescence+staining.">Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.</a> Biol Proced Online. 17, (2015).</li><li>Wang, 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</li></ol>

作者没有什么可透露的。

IST 结合 IHC 提供了一个基本的工具, 以检测, 定性和量化抗原特异性 CD8 T 细胞在本机环境与其他细胞和组织结构的上下文。在这里, 我们描述了 IST 结合 IHC 的详细程序, 其次是定量图像分析, 以确定的位置, 丰度和表型的抗原特异性 CD8 T 细胞在淋巴结的恒河猴。类似的染色可以适用于人类, 老鼠, 或其他物种组织的 MHC I 体是可用的。此外, 肽 MHC 类 II 聚丙烯或 dextramer 染色可以使用相对相似的方法来?...

T 细胞对许多免疫过程至关重要, 包括检测和消除病毒感染的细胞, 预防自身免疫, 协助 B 细胞和血浆细胞产生抗体, 以及检测和消除癌细胞。多肽/MHC I. 类抗原特异性 CD8 t 细胞聚丙烯染色的研究进展1和最近发展的 mhc 类 II 聚丙烯染色 CD4 t 细胞2流式细胞术彻底改变了我们的学习和理解能力T 细胞的免疫。流式细胞仪在确定抗原特异性 t 细胞的数量和表型时非常有用, 但不允许检测抗原特异性 t 细胞在组织中的其他细胞和结构的空间定位, 以及当前用于提取流式细胞术所需 T 细胞的分类技术在非组织中的有效性有限3。
我们和其他人已经开发的方法使用肽负载 MHC i 类和 II 类聚丙烯或 multimer 试剂染色抗原特异性 CD8 和 CD4 T 细胞在组织中4,5,6,7,8,9,10,11,12,13. 这些 IST 方法可以确定组织中抗原特异的 CD8 和 CD4 T 细胞的位置、丰度和表型, 并提供一种方法来检测这些细胞与组织中其他细胞和结构的关系。我们的小组广泛使用 MHC I 聚丙烯染色研究人体免疫缺陷病毒 (HIV) 和猴免疫缺陷病毒 (SIV) 特定的 CD8 T 细胞的淋巴, 生殖器和直肠组织获得了解艾滋病毒和 SIV 免疫并识别成功接种策略的相关性14,15,16,17。此外, 我们还开发了一种技术, 结合 IST 与原位杂交 (一), 以本地化和量化病毒特异性 CD8 T 细胞和病毒感染的细胞在组织和确定的在体内effector-to-target 水平18,19。
在这里, 我们描述了一个协议使用多肽负载 MHC I 体染色抗原特异性 CD8 T 细胞在新鲜的组织切片, counterstain 组织使用 IHC, 并量化细胞与特定的表型在特定的组织舱室。这些过程与我们最近出版的李et al.所使用的程序相同, 在该出版物中, 我们确定了在猕猴慢性 siv 感染过程中, 在淋巴组织中的 siv 特异性 T 细胞的位置、丰度和表型,20。
在这个过程中, 新鲜的组织被切片和培养过夜与肽负载 MHC I 体共轭的荧光素硫氰酸盐分子 (FITC)。然后固定在甲醛。在修复组织后, 从 MHC 体的信号被放大使用兔抗 FITC 抗体和孵化与荧光标记兔 IgG 抗体, 进一步放大信号从绑定体。IHC 与 IST 结合用于抗原特异 T 细胞和周围细胞的特征。抗体, 承认表位在细胞表面或在胞外空间是包括在初级孵化与体。识别细胞内抗原表位的抗体需要在染色前渗透细胞壁。使用共聚焦显微镜对染色组织切片进行成像, 并使用共焦软件进行分析。标记细胞使用共聚焦显微镜软件或 ImageJ 进行量化。所述的协议可用于在任何有 MHC I 体的组织中基本上染色任何抗原特异的 CD8 T 细胞。

<table align="center" border="1" cellpadding="0" cellspacing="0" style="width:906px;" width="906">
	<tbody>
		<tr>
			<td>MHC-I monomer</td>
			<td>NIH tetramer core facility</td>
			<td></td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>ExtrAvidin-FITC</td>
			<td>Sigma-Aldrich</td>
			<td>E2716</td>
			<td>Materials for MHC-tetramer preparation</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBL6391V</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>J.T.Baker</td>
			<td>4204-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol/gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>SLBH2672V</td>
			<td></td>
		</tr>
		<tr>
			<td>n-propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>rat-a-h-CD8 (1:500)</td>
			<td>Acris</td>
			<td>0714</td>
			<td>Antibody unstable, use single use frozen aliquot</td>
		</tr>
		<tr>
			<td>m-a-h-CD20 (1:500)</td>
			<td>NOVOCASTRA</td>
			<td>6026819</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-h-Ki67 (1:500)</td>
			<td>Vector</td>
			<td>6022201</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-m-A488 (1:2000)</td>
			<td>Jackson Immunoresearch</td>
			<td>124083</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rb-Cy3 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>106232</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-rat-Cy5 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>118088</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-a-h-IgM-Dylight649 (1:5000)</td>
			<td>Jackson Immunoresearch</td>
			<td>86579</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresstome: VF-300 Microtome</td>
			<td>Precisionary Instruments, LLC</td>
			<td>1079</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Set Instant Adhesive</td>
			<td>Loctite</td>
			<td>46551</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well flat bottomed tissue culture plates</td>
			<td>Falcon</td>
			<td>353226</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Globe scienfitic Inc.</td>
			<td>#1321</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor&#160; blade</td>
			<td>Ted Pella, Inc</td>
			<td>121-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Disposable Scalpel</td>
			<td>FEATHER SAFETY RAZOR CO. LTD.</td>
			<td>No. 21</td>
			<td></td>
		</tr>
		<tr>
			<td>Round paintbrush #2</td>
			<td>PRINCETON ART &#38; BRUSH CO.</td>
			<td>4350R</td>
			<td>Can trim as needed with razor</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW_Viewer4.0</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues

T 细胞对许多免疫过程至关重要, 包括检测和消除病毒感染的细胞, 预防自身免疫, 协助 B 细胞和血浆细胞产生抗体, 以及检测和消除癌细胞。流式细胞术分析抗原特异性 t 细胞 MHC-聚丙烯染色的进展, 改变了我们研究和理解 t 细胞免疫的能力。流式细胞仪在确定抗原特异性 t 细胞的数量和表型时非常有用, 但不能确定抗原特异性 t 细胞在组织中的其他细胞和结构的空间定位, 以及当前的分类技术提取流式细胞术所需的 T 细胞在非组织中的有效性有限。原位MHC-聚丙烯染色 (IST) 是一种技术的可视化 T 细胞是特定的抗原感兴趣的组织。结合免疫组化 (IHC), IST 可以确定抗原特异性 CD8 和 CD4 T 细胞在组织中的丰度、位置和表型。在这里, 我们描述一个协议, 以染色和列举抗原特异的 CD8 T 细胞, 与特定的表型位于特定的组织隔间。这些程序与我们在最近出版的李et al.中使用的相同, 标题是 "在卵泡中的猿猴免疫缺陷病毒产生细胞在体内被 CD8 的+细胞部分抑制."所描述的方法是广泛适用的, 因为它们可以用于本地化, 表型, 并量化基本上任何抗原特异性 CD8 T 细胞的 MHC 体是可用的, 在任何组织。

图 1显示了如何使用共聚焦显微镜收集共聚焦图像。图 2演示使用 ImageJ 进行定量图像分析。图 3和4显示了来自 SIV 感染的恒河猴的淋巴结组织的代表性图像, 体、CD8 抗体和 CD20 抗体染色, 并有助于证明 mhc-聚丙烯染色的特异性。图 3比较 mhc 类 i 体载有一个多肽从 SIV 相比, 从同一组?...

Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

在这里, 我们描述一个方法, 结合原位MHC 聚丙烯染色与免疫组化, 以确定的定位, 表型和数量的抗原特异性 T 细胞在组织。本协议用于确定抗原特异性 CD8 T 细胞相对于其他细胞类型和组织结构的空间和表型特征。

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

in-situ-mhc-tetramer-staining-quantitative-analysis-to-determine

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

12.2K 次观看.  University of Minnesota. 在这里, 我们描述一个方法, 结合原位MHC 聚丙烯染色与免疫组化, 以确定的定位, 表型和数量的抗原特异性 T 细胞在组织。本协议用于确定抗原特异性 CD8 T 细胞相对于其他细胞类型和组织结构的空间和表型特征。

视频: In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

代谢图谱: 定量酶化学和组织化学法测定脱氢酶在细胞和器官中的活性

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

摘要

探索更多视频

此视频中的章节