普拉莫德Nehete博士和夫人Bharti公司Nehete猕猴血液和PBMC样品;部分支持这项工作是由美国国立卫生研究院授予的AI 46969资金，所有的细胞培养基，由中央媒体实验室，这是由美国国立卫生研究院授予CA资助16672。

总额以及记忆T细胞子集的表型和功能的详细分析的程序可分为四个部分：1）细胞的制备及抗原刺激，2）表面标志物染色，3）细胞内细胞因子染色和4）流式细胞仪分析。 1。细胞的制备及抗原刺激<ol><li>既新鲜分离以及冷冻保存外周血单个核细胞（PBMC）的，在此协议使用。肝素或枸橼酸恒河猴静脉血液样本中分离的外周血单核细胞密度梯度沉淀，使用聚蔗糖- Hypaque（Histopaquen - 1077，Sigma - Aldrich公司，圣路易斯，密苏里州）。存储在90％FCS的PBMC等分10％，在液氮冷冻和二甲基亚砜。 </li><li>使用冷冻保存外周血单个核细胞时，从液氮冷冻PBMC瓶在37℃水浴中迅速解冻，轻轻混合，用RPMI - 1640（Hyclone公司实验室，洛根，UT）洗去冻结中期和完整的媒体重新悬浮厘米; RPMI - 1640具有10％的补充热灭活FCS（Hyclone公司实验室），2mm的L -谷氨酰胺（Sigma - Aldrich公司），100U/ml青霉素/链霉素（Invitrogen公司），并于6培养 - 以及组织培养板在37℃过夜彗星在饱和湿度5％的CO 2气氛。第二天早晨，活细胞计数测定台盼蓝染料排斥法和重新悬浮在CM。 </li><li>细胞治疗非特异性与抗原特异性刺激因子分析其次是不同的：（一）非特异性刺激与佛波醇-12 - 肉豆蔻酯（PMA）和离子霉素（一）（Sigma - Aldrich公司，圣路易斯，MO），分装在体积的0.1毫升细胞（1.0 × 10 6细胞/孔）接种在96孔组织培养板（BD公司），新泽西州富兰克林湖，个别井。 PMA和离子霉素，分别用于在终浓度为50ng/ml和500ng/ml。 96孔组织培养板的最终总量0.2ml/well：（B）抗原特异性刺激，1.0 × 10 6卷1.0毫升的细胞在24孔组织培养板中的个别井（BD公司镀，新泽西州富兰克林湖），井前视为与修改9。简言之，在24孔组织培养板涂有2.5微克/毫升/以及羊抗鼠IgG（H + L）（克尔凯郭尔和佩里实验室，马里兰州盖瑟斯堡）在50毫米的Tris溶液（PH值8.6），4 ° C过夜。第二天早上板块洗净，用无菌PBS和协同刺激单克隆抗体的表达CD49d，克隆9F10（BD Biosciences公司;加利福尼亚州圣何塞）两次，分别加入10微克/毫升/以及随后一小时的孵化，在37 ° C。孵化后，用无菌PBS洗涤板在室温下的两倍。利益抗原（包括6个HIV包膜肽 8鸡尾酒添加的每个肽的终浓度为10μg/ml。附加井仅在CM细胞作为阴性对照准备最后的总体积为每孔24孔组织培养板为1.0 ml。 </li><li>细胞培养6小时，在37 ° C的饱和湿度5％的CO 2气氛。 Brefeldin一（Sigma - Aldrich公司，圣路易斯，密苏里州）在10μg/ml的文化，为最后的4.5小时的刺激。随后，细胞被转移到5毫升聚丙烯管洗净，用冷（4 °彗星）流洗缓冲液（贝科的PBS（，钙2 /镁2 -免费DPBS Life技术“，罗克维尔，医师）用2％热灭活FCS），然后与不同的荧光标记抗体染色处理。 </li></ol> 2。免疫表面标志物的染色<ol><li> 1ul活/死可以解决旱厕荧光活性染料染色刺激细胞在4 ° C的黑暗30分钟，洗后用冷水流缓冲液洗一次。 </li><li>然后，下面的适当滴定，荧光标记的单克隆抗体细胞：抗CD3 PE - Cy7（SP34 - 2），抗- CD8 Alexa700（RPA - T8），抗CD28 PerCP cy5.5（L293）抗CD95的APC（DX2）和抗CD4太平洋蓝（OKT4），所有自BD Biosciences公司（新泽西州富兰克林湖，），除CD4 +，太平洋蓝（OKT4）从eBioscience公司（加利福尼亚州圣迭戈）。细胞孵育30分钟，在4 ° C，在黑暗中。 </li><li>对于每个实验10,11利用补偿控制和荧光减一（FMO）的控制。利用补偿控制，以确定每一个人的染色到其他探测器的外溢coeffecients。补偿控制，一套完整的含有猴细胞悬浮液管与荧光标记的单克隆抗体染色，单独作为单一颜色的污渍。鱼类统营处是一个多色染色的组合，包含所有的试剂，而是一个利益，是用来确定之间的边界通过复制自体荧光的水平和数据的正面和负面的人口目前在完全染色的样本。 </li><li>染色的细胞，然后用冷水流缓冲液洗的洗，和固定在固定/通透的解决方案（BD Biosciences公司;加利福尼亚州圣何塞）（在这个阶段的细胞，可存储在一夜之间在4 ° C黑暗环境中至少10分钟）然后进行细胞内细胞因子染色。 </li></ol> 3。细胞内细胞因子染色<ol><li>固定与流动缓冲液洗洗涤细胞，然后烫/洗涤液（BD Biosciences公司;加利福尼亚州圣何塞）是根据制造商的说明补充。简单地说，细胞培养1X烫/缓冲液洗为10分钟，在4 ° C黑暗环境中0.1毫升。 </li><li>随后，细胞培养与适当滴定的FITC标记的抗干扰素γ（B27）和PE标记的抗IL - 2为60分钟（MQ1 - 17H12）4 ° C黑暗环境中。 </li><li>那么细胞洗两次与通透的解决方案</li><li>最后一次洗涤后，将细胞重新悬浮在1％多聚甲醛在DPBS和24小时内遭受流式细胞仪分析。 </li></ol> 4。流式细胞仪分析<ol><li>染色的细胞获得一个LSR II流式细胞仪（BD公司，加利福尼亚州圣何塞）或青色ADP（DAKO，Carpinteria的CA）。通过使用FlowJo软件（树星，亚什兰，或），流式细胞仪数据分析。 </li><li>图。 1显示了具有代表性的动物，从不同的T细胞亚群的分析利用浇注计划。首先通过前向散射（FSC）与侧散射（SSC），门控的淋巴细胞，再活的淋巴细胞被确定基于SSC和水产人口负。 T细胞，然后验明正身由检测的CD4 + CD8 -（CD4 + T细胞），CD3 +表达和CD4 - CD8 +（CD8 + T细胞）的种群内的CD3 + T细胞的人口。的CD4 +和CD8 + T细胞被进一步区分到不同的子天真（TN，CD28 + CD95 -），中央内存（TCM，CD28 + CD95 +）和效应记忆体（TEM，CD28 - CD95 +）细胞为CD28和CD95表达的基础上在文学5日，10所述。 </li><li>图。 2如何CD4 +和CD8 + T细胞不同亚群细胞因子的产生，在刺激与PMA和离子霉素（PMA + I）（IFN -γ和/或IL - 2）功能的能力进行评估。 </li></ol> 5。代表流式细胞仪数据恒河猴外周血单个核细胞组成，使用流式细胞仪分析来确定。图1显示了门控计划为代表的动物从一个不同的T细胞亚群的分析利用。首先通过前向散射（FSC）与侧散射（SSC），门控的淋巴细胞，再活的淋巴细胞被确定基于SSC和水产人口负。 CD3 +表达的T细胞，然后确定。 CD4 + CD8 -（CD4 + T细胞）和CD4 - CD8 +（CD8 + T细胞）的种群内的CD3 + T细胞的人口也决心。图2所示的CD4 +和CD8 + T细胞进一步区分到不同的子集的CD28和CD95表达的幼稚（TN，CD28 + CD95 -），中央内存（TCM，CD28 + CD95 +）和效应记忆体（TEM的基础上， CD28 - CD95 +）细胞。图2A显示IFN -γ和IL - 2在未刺激和PMA /离子霉素刺激CD4 + T细胞及亚群，和图2B生产显示的INF -γ和IL - 2在未刺激和PMA /离子霉素刺激CD8 + T细胞的生产，和亚群注意：在每个子集，不同的细胞因子刺激引起的。 <a href="https://www.jove.com/files/ftp_upload/1743/1743fig1.jpg"><img src="/files/ftp_upload/1743/1743fig1tmb.jpg" alt="图1" border="0" /></a> 图1。浇注流流式细胞仪分析外周血单个核细胞组成的战略在恒河猴macqaues <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2a.jpg"><img src="/files/ftp_upload/1743/1743fig2atmb.jpg" alt="图2a" border="0" /></a> <a href="https://www.jove.com/files/ftp_upload/1743/1743fig2b.jpg"><img src="/files/ftp_upload/1743/1743fig2btmb.jpg" alt="图2b" border="0" /></a> 图2。 （一）总的CD4 + T细胞亚群。细胞因子生产概况（二）总CD8 + T细胞及亚群细胞因子的生产概况。

<ol>
	<li>Ogg, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+HIV-1-specific+cytotoxic+T+lymphocytes+and+plasma+load+of+viral+RNA.">Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.</a> Science. 279, 2103-2106 (1998).</li><li>Borrow, P., Lewicki, H., Hahn, B. H., Shaw, G. M., Oldstone, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-specific+CD8++cytotoxic+T-lymphocyte+activity+associated+with+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+infection.">Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.</a> J Virol. 68, 6103-6110 (1994).</li><li>Koup, 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=Temporal+association+of+cellular+immune+responses+with+the+initial+control+of+viremia+in+primary+human+immunodeficiency+virus+type+1+syndrome.">Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.</a> J Virol. 68, 4650-4655 (1994).</li><li>Walker, J. M., Maecker, H. T., Maino, V. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+analysis+in+SIV-infected+rhesus+macaque.">Multicolor flow cytometric analysis in SIV-infected rhesus macaque.</a> Methods Cell Biol. 75, 535-557 (2004).</li><li>Pitcher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+homeostasis+of+T+cell+memory+in+rhesus+macaque.">Development and homeostasis of T cell memory in rhesus macaque.</a> J Immunol. 168, 29-43 (2002).</li><li>Jung, T., Schauer, U., Heusser, C., Neumann, C., Rieger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+intracellular+cytokines+by+flow+cytometry.">Detection of intracellular cytokines by flow cytometry.</a> J Immunol Methods. 159, 197-207 (1993).</li><li>Keeney, T. S., Nomura, L. E., Maecker, H. T., Sastry, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+macaque+whole+blood+for+antigen-specific+intracellular+cytokine+production+by+T+lymphocytes.">Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes.</a> J Med Primatol. 32, 23-30 (2003).</li><li>Gauduin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intracellular+cytokine+staining+for+the+quantitation+of+antigen-specific+CD4++T+cell+responses+in+rhesus+macaques.">Optimization of intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.</a> J Immunol Methods. 288, 61-79 (2004).</li><li>Baumgarth, N., Roederer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+approach+to+multicolor+flow+cytometry+for+immunophenotyping.">A practical approach to multicolor flow cytometry for immunophenotyping.</a> J Immunol Methods. 243, 77-97 (2000).</li><li>De Rosa, S. C., Herzenberg, L. A., Roederer, M. 1. 1. -. c. o. l. o. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13-parameter+flow+cytometry:+identification+of+human+naive+T+cells+by+phenotype,+function,+and+T-cell+receptor+diversity.">13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.</a> Nat Med. 7, 245-248 (2001).</li><li>Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+fluorescence+compensation+and+visualization+of+FACS+data.">New approaches to fluorescence compensation and visualization of FACS data.</a> Clin Immunol. 110, 277-283 (2004).</li></ol>

解决方案和缓冲器<ul><li>聚蔗糖- Hypaque（Histopaquen - 1077，Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li> RPMI - 1640培养基（Hyclone公司实验室，洛根，UT） </li><li>胎牛血清（FBS）（Hyclone公司实验室，洛根，UT） </li><li> L -谷氨酰胺（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>青霉素/链霉素（Invitrogen公司;卡尔斯巴德，CA） </li><li>台盼蓝试剂（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>的50 mM的Tris，PH值8.6，过滤和储存在4 ° C </li><li>贝科的PBS（DPBS，Ca2/Mg2-free; Life技术，马里兰州洛克维尔） </li><li>流洗涤缓冲液：含2％FBS DPBS补充，储存于4 ° C </li><li> BD Cytofix / Cytoperm固定/通透解决方案套件（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>激活剂<ul><li>佛波醇-12 - 肉豆蔻13醋酸乙烯酯（PMA）（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>离子霉素（Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>共刺激表达CD49d单克隆抗体，克隆9F10（BD Biosciences公司;加利福尼亚州圣何塞） </li><li>亲和纯化抗体的F（ab）2片段的羊抗鼠IgG（H + L）（克尔凯郭尔和佩里实验室，马里兰州盖瑟斯堡） </li><li>特定的激活剂可以包括肽池，单肽，或整个蛋白质</li></ul>细胞因子的分泌抑制剂<ul><li> Brefedin一个（博鳌亚洲论坛，Sigma - Aldrich公司，圣路易斯，密苏里州） </li><li>高尔基的插头，可交替使用（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>抗体<ul><li>活/死可以解决旱厕荧光染色试剂盒（Invitrogen公司;卡尔斯巴德，CA） </li><li> CD3 + PE - Cy7（SP34 - 2），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD4太平洋蓝（OKT4），（eBiosciences（加利福尼亚州圣迭戈） </li><li> CD8 + Alexa700（RPA - T8），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD28 PerCP cy5.5（L293），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> CD95的APC（DX2），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li>干扰素- FITC（B27），（BD Biosciences公司;加利福尼亚州圣何塞） </li><li> IL - 2的PE（MQ1 - 17H12），（BD Biosciences公司;加利福尼亚州圣何塞） </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

猕猴模型是目前最好的艾滋病毒和艾滋病的模型，了解其发病机制，以及疫苗和疗法的发展<sup&gt; 1,2,3</sup&gt;。在这里，我们描述了一个详细的表型和功能分析的细胞免疫反应，特别是细胞内的CD4细胞因子生产+和CD8 + T细胞以及个人记忆亚群的方法。我们精确的定量和定性的措施，为生产干扰素γ（INF -γ）和白细胞介素（IL）-2获得的CD4 +和CD8 + T细胞从PMA加离子霉素（PMA + I）刺激的恒河猴PBMC的。细胞因子在记忆细胞不同亚群的不同。此外，该协议为我们提供的灵敏度，证明即使是轻微的组分抗原特异性CD4 +和CD8 + T细胞内的PBMC样本在我们的实验室开发的HIV包膜肽鸡尾酒疫苗免疫恒河猴的子集。多色流式细胞术是一种强大的工具，准确地识别不同人群的T细胞<sup&gt; 4,5</sup&gt;与细胞因子产生能力<sup&gt; 6</sup&gt;非特异性或抗原特异性刺激以下<sup&gt; 5,7</sup&gt;。

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

我们展示了总额以及记忆CD4 +和CD8 + T细胞在猕猴，艾滋病毒/艾滋病疫苗研究的理想模型的子集的详细的表型和功能特性的多色流式细胞仪的实用。

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

multicolor-flow-cytometry-analyses-cellular-immune-response-rhesus

Research

JoVE Journal

Immunology and Infection

21.0K 次观看.  MD Anderson Cancer Center - University of Texas. 我们展示了总额以及记忆CD4 +和CD8 + T细胞在猕猴，艾滋病毒/艾滋病疫苗研究的理想模型的子集的详细的表型和功能特性的多色流式细胞仪的实用。

视频: Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.