1。分离人CD4 +从外周血T细胞（见图1） <ol><li>收集到真空采血管外周静脉血（40至60毫升） 管含肝素钠（ 流式细胞迪肯森）。 </li><li>外周血中，然后转移的真空采血管管50毫升聚苯乙烯锥形管（每管的血液20毫升）。 </li><li> RosetteSep CD4 + T细胞的隔离剂（干细胞技术）是在1 mL体积不超过20毫升血液50毫升管和混合添加。 </li><li>然后将混合物在20-25℃孵育20分钟。 </li><li>后20分钟，钙和镁（CMF）磷酸盐缓冲液PBS（Hyclone公司 ] W / 2％热灭活（56℃30分钟）小牛血清[FBS（Hyclone公司 ）]添加到混合物以实现最终体积为40 mL。 </li><li>稀释混合物（30毫升）15毫升淋巴细胞分离液上，然后分层[LSM（Cellgro）]新鲜聚苯乙烯50毫升锥形管。 </li><li> 50毫升管，然后离心1200 × 克为20分钟关闭制动。 </li><li> 50 mL管从离心机仔细，使LSM和媒介之间的接口，是不是感到不安。在界面上的细胞收集在10 mL吸管和食入50毫升聚苯乙烯的锥形管放置。 </li><li>收集到的接口，用PBS W / 2％FBS稀释到终体积为50毫升。 </li><li>含有50毫升稀释后的接口管，离心1200 × 克为10分钟制动。 </li><li>离心后，上清吸气和颗粒悬浮在10毫升的PBS瓦特/ 2％FBS。 </li><li>细胞悬浮液，然后合并成一个单一的50 mL锥形管，用PBS W / 2％FBS稀释至50毫升。 </li><li>管中结合细胞悬液，离心10分钟，以除去所有剩余的LSM 300 × 克 。 </li><li>上清吸入的颗粒，然后悬浮在10 MLS的RPMI - 1640培养基（Hyclone公司 ），含10％胎牛血清，100 U / ml的青霉素，链霉素100微克/毫升（Hyclone公司）和2毫米L -谷氨酰胺（ CellGro ）（完整的RPMI -），并使用一个血球计算。 </li><li>纯化的CD4 + T细胞的浓度，然后调整到 3X10 6 /毫升用RPMI完成。 </li><li> CD4 +&#39;，然后刺激他们培养anti-CD3/anti-CD28再加以珠[T细胞扩展套件（ 美天旎生物技术 ）]三珠每个细胞比例72小时在37℃5％CO 2饱和湿度彗星孵化器。 </li></ol> 2。感染的CD4 + T细胞与HI​​V - 1（见图1） <ol><li>含有刺激的CD4 + T细胞的细胞悬液从培养板，并用一个血球计数。 </li><li> CD4 + T细胞（约2X10 6）添加到15毫升锥形管作为未受控制使用。 </li><li>其余的CD4 +的T -细胞悬液放入50 mL锥形管感染。 </li><li>包含的CD4 + T细胞悬浮液在300 × 克离心10分钟和培养上清管吸出。 </li><li>刺激的CD4 + T细胞是直接重悬在文化含有病毒颗粒的液体，然后在1200 × 2小时，在20-25℃9克分拆感染。通常情况下，我们感染复制缺乏艾滋病毒株（例如，DHIV - 3）或0.01一个教学语言的一个复制主管病毒的CD4 + T细胞在一个受感染的多重5（MOI），（如艾滋病毒，1 NL4 / 3） 。最终成交量是不重要的，但必须平衡离心管。凝聚胺（ 圣克鲁斯 ）被添加到培养液之前spinfection在终浓度为10微克/毫升。 </li><li> spinfection完成后，上清和细胞悬浮在一个完整的RPMI - 3X10 6 / mL的细胞密度辅以100 U / mL的IL - 2。感染和未受感染的细胞，如果一个不称职的病毒复制被用来感染的细胞或如果复制主管病毒感染细胞的5-7天72小时的文化。媒体应改为每48小时期间文化。 </li></ol> 3。人类外周血自然杀伤细胞的分离<ol><li>收集从隔离到真空采血管的CD4 + T细胞相同的捐助外周静脉血（〜100毫升） 管含肝素钠（ 流式细胞迪肯森 ）。 </li><li>从真空采血管的血液，然后转移到50毫升20毫升分装在锥形管的管。 </li><li>在真空采血管管，然后洗净，用CMF的汉克斯平衡盐溶液的HBSS（Hyclone公司 ）的8 mL和清洗都转移到50 mL的conicals containi吴的20毫升的外周血。每50 mL锥形量是35毫升。 </li><li>稀释血液15毫升的LSM在一个新的50 mL锥形管和400 × 克离心30分钟关闭制动管上，然后分层。 </li><li>从离心管小心取出，LSM和媒介之间的接口是收获从每个使用10毫升吸管管，食入50 ml锥形管。 </li><li> CMF的HBSS（400 × 克离心15分钟的制动），细胞悬浮液洗一次。 </li><li>后，先洗上清拆除和由此产生的颗粒结合成一个单一的50 mL锥形和CMF方案的HBSS（30 X克离心10分钟的制动）洗净，用两次删除所有剩余的LSM。 </li><li>最后一次洗涤后的沉淀是resupended ACK裂解液10毫升（150毫米NH 4 CL KHCO 3，1.0毫米和0.1毫米EDTA pH值7.3），并在室温（20-25℃）孵育5分钟。这一步是必要的，以消除从外周血单个核细胞（PBMC）的污染红细胞。 </li><li> 10分钟后。 40毫升的CMF的PBS加入细胞悬液，以阻止裂解反应。 </li><li> 10分钟的制动管与细胞悬浮PBMC和裂解红细胞，然后离心300 × 克 。 </li><li>除去上清液，5毫升的PBS悬浮颗粒与2％FBS和2毫米EDTA和计算使用一个血球。 </li><li>自然杀伤细胞（NK），然后从外周血单个核细胞阴性选择的人（ 美天旎生物技术公司 ）根据制造商的说明使用的NK细胞分离试剂盒分离。 </li><li>列流动收集15毫升管和10分钟的制动管离心300 × 克 。 </li><li>上清液被删除和悬浮颗粒。单管的悬浮颗粒结合在一个的1 Iscove的修改贝科的中等[IMDM（GIBCO）]毫升总体积辅以10％AB +热灭活（56 °彗星30分钟）人类血清（HS）和与计hemacytometer。 </li><li>细胞悬液的体积IMDM w/10％房协与调整，最终使NK细胞的密度是3X10 6 /毫升。将细胞悬液分成两半。 NK细胞接收200 U / mL的重组人白细胞介素-2。无细胞因子治疗，另一组则留在细胞介质。 </li><li> NK细胞培养，然后在37℃，5％的CO 2孵育过夜</li><li>由此产生的NK细胞，然后作为一个CD107a脱粒检测或51铬释放法检测效应细胞。 </li></ol> 4。 HIV - 1感染细胞的分离纯化（见图1） <ol><li>感染细胞的培养液中含有从板块中删除， 并在300 X克离心10分钟的制动。 </li><li>上清吸气和2％FBS和2毫米EDTA和一个血球计数细胞，颗粒悬浮在2毫升的PBS。 </li><li>不包含HIV - 1感染的细胞是孤立的，从海港病毒所利用的事实，HIV - 1向下调节CD4 +。对于这样做的目的CD4阳性分离试剂盒（Invitrogen公司）是根据异常的制造商，一个每个细胞比CD4珠是用来指示使用。 </li><li>拆除的CD4 + T细胞后，死细胞从细胞纯化窝藏病毒的人口，根据制造商的说明使用死细胞去除试剂盒（美天旎生物技术公司）。 </li><li>离心300 × 克列中的死细胞去除后，通过流量为10分钟制动。上清被删除，颗粒悬浮于1毫升的RPMI完成细胞计数。 </li><li>感染细胞的纯化，现在准备在CD107a脱粒检测或51铬释放法的目标。 </li></ol> 5。 CD107a脱颗粒含量（见图2） <ol><li>从板块中删除NK细胞培养和细胞悬液，计数。 </li><li> NK细胞悬液，离心300 × 克为10分钟制动。 </li><li> NK细胞是悬浮的RPMI完成和终浓度调整到10 6细胞/ ml。 </li><li>从隔离在上面的步骤产生的感染和未感染T细胞的细胞悬浮液离心300 ×克为10分钟制动。 </li><li>上清被删除，颗粒悬浮在2 × 10 6细胞/ mL的RPMI完成。 </li><li> K562细胞作为阳性对照靶细胞，因为它们很容易受到NK细胞裂解。 T他的细胞在300 × 克离心10分钟的制动，上清被删除的RPMI完成重新悬浮颗粒在2 × 10 6细胞/ mL </li><li> 96 - V -见底孔板100个目标和100 MCL效应的MCL被添加到井。 </li><li>良好含有100 mcLof效应和中期的100 MCL无靶细胞，将用于非特异性NK细胞脱颗粒。 </li><li>每口井10 mcLFITC共轭反CD107a（BDIS）。 </li><li>该板块，然后离心20 × 克为2分钟制动。 </li><li>离心板在37 ° C和5％的CO 2孵育4小时。 </li><li>孵化后5分钟的制动板是离心400 × 克 。 </li><li>上清液从沉淀。 </li><li>每口井，然后冰20分钟以下的面板与荧光标记的抗体染色，在总共100 mcLof流缓冲液（PBS W / 2％FBS和0.1％，为NaN 3） <ol><li> anti-CD3/14/20 [太平洋蓝共轭（Biolegend） ] </li><li>抗CD56 + [APC（Biolegend）] </li></ol></li><li>经过20分钟的潜伏期，200流缓冲区的MCL，每孔加入离心5分钟的制动板是400 ×克。 </li><li>流缓冲区然后小心地从每口井用2毫升吸吸管装200 MCL micropipet提示，同时确保不打扰颗粒吸气。 </li><li> 15日和16步重复一次。 </li><li>颗粒悬浮于200 1％多聚甲醛在CMF的PBS MCL。细胞悬浮液，然后转移到流管。 </li><li>总成交量为300 MCL加入1％多聚甲醛在CMF的PBS〜100 MCL，每管。 </li><li>收集的事件（2X10 4）相应的NK细胞，使用流式细胞仪DIVA（BDIS）收购的软件和分析使用流量乔软件 （TreeStar）由流动流式细胞仪。 </li></ol> 6。 CD107a浇注战略（见图3） <ol><li>单细胞的大门使用的前向散射高度（FSC - H）和前向散射宽度（FSC - W）情节。 </li><li>对于大小与粒度情节上创建一个FSC VS侧散射（SSC）情节单细胞门。通常情况下，淋巴细胞有相对较低的粒度和介质细胞的大小。 </li><li>淋巴细胞门的细胞，然后在CD3/14/20染色细胞与NK细胞CD56的染色细胞向门点图绘制，而忽略单核细胞（CD14），T细胞（CD3 +）和B细胞（ CD20的）。 </li><li> CD56的POS门人口，然后评估CD107a 。 </li><li>无目标组的NK细胞是用来设置什么被认为是CD107a负的大门。 </li></ol> 51铬释放法（见图4）7。 <ol><li>被感染的靶细胞标记共500 MCL 125 MCCI 51 班（Perkin Elmer公司）在saline/10 2小时6细胞在37 ° C和5％的CO 2。 10 6 K562细胞作为阳性对照标记与100 MCCI 51班共500 MCL为2小时37 ° C和5％的CO 2。总量是500 MCL用RPMI完成。 51卷训练班添加到靶细胞计算:（http://las.perkinelmer.com/Catalog/RadCalculator.htm?Mode=RadioactivityCalculator） </li><li>以前孤立的NK细胞的靶细胞标记，从文化和计数。 NK细胞的细胞培养物中含有与刹车系统在10分钟离心300 ×克。 </li><li> NK细胞的小球含有悬浮的RPMI完成，并分成3管。一管，2.5 × 10 5 / ml的第二管和5X10 5 /毫升第三管中，细胞密度调整为10 5 /毫升。 </li><li>标记目标细胞是从孵化器中删除，并添加到每管4.5毫升的RPMI完成。 300 × GF分钟或10分钟，然后离心管。 </li><li>上清浇灭了，小心不要打扰到放射性液体废物的容器的沉淀。 </li><li>重复步骤4和5的两倍，删除所有未吸收的 51班。第三洗上清可处置的非放射性液体废物的容器中。 </li><li>标记的目标，然后再悬浮的RPMI完成最终细胞密度的10 5 /毫升。 </li><li> 100 MCL的标记目标是分装成井的最后一个10 4靶细胞的细胞数/ 96 - V -底部孔板。 </li><li>表1是一个典型的设置为51铬释放法的例子。每个组都做了一式三份。 <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><tD&gt; 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td></tr><tr><td>一</td><td> K562 E:T（1:1） </td><td> K562 E:T（1:1） </td><td> K562 E:T（1:1） </td><td> K562 E:T（2.5:1） </td><td> K562 E:T（2.5:1） </td><td> K562 E:T（2.5:1） </td><td> K562 E:T（5:1） </td><td> K562 E:T（5:1） </td><td> K562 E:T（5:1） </td></tr><tr><td>乙</td><td> UI E:T（1:1） </td><td> UI E:T（1:1） </td><td> UI E:T（1:1） </td><td> UI E:T（2.5:1） </td><td> UI E:T（2.5:1） </td><td> UI E:T（2.5:1） </td><td> UI E:T（5:1） </td><td> UI E:T（5:1） </td><td> UI E:T（5:1） </td></tr><tr><td>彗星</td><td> HIV - 1感染E:T（1:1） </td><td> HIV - 1感染E:T（1:1） </td><td> HIV - 1感染E:T（1:1） </td><td> HIV - 1感染E:T（2.5:1） </td><td> HIV - 1感染E:T（2.5:1） </td><td> HIV - 1感染E:T（2.5:1） </td><td> HIV - 1感染E:T（1:1） </td><td> HIV - 1感染E:T（1:1） </td><td> HIV - 1感染E:T（1:1） </td></tr><tr><td> ð </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Ë </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td> K562细胞自发释放</td><td> K562细胞自发释放</td><td> K562细胞自发释放</td><td>用户界面自发发布</td><td>用户界面自发发布</td><td>用户界面自发发布</td><td> HIV - 1感染自发释放</td><td> HIV - 1感染自发释放</td><td> HIV - 1感染自发释放</td></tr><tr><td>摹</td><td> K562最大释放</td><td> K562最大释放</td><td> K562最大释放</td><td> UI最大释放</td><td> UI最大释放</td><td> UI最大释放</td><td> HIV - 1感染Maxmum发行</td><td> HIV - 1感染Maxmum发行</td><td> HIV - 1感染Maxmum发行</td></tr></tbody></table></li><li> 100 MCL上述NK细胞悬液加入到每孔含有的靶细胞。 </li><li> 100 MCL的目标是加入到相应的效应细胞是最大的和自发释放组缺席的井。自发释放组孵育增加100 MCL的RPMI完成。 </li><li> 96孔板离心20 × 克为2分钟制动。 </li><li>离心盘，然后在37℃孵育° C和5％CO 2 4小时。 </li><li>经过4小时的潜伏期5 MCL的一个人体红细胞的RPMI完成1:10稀释，每孔加入除最大限度地释放井。 </li><li> 100 MCL是10％的十二烷基硫酸钠添加到最大限度地释放井。 </li><li>该板块是400 × 克离心5分钟沉淀细胞。 </li><li> 100无细胞上清液MCL是收获的每口井，并放入单独的γ-计数管（Perkin Elmer公司 ） 。管被放置在一个2470自动伽马计数器（Perkin Elmer公司 ）。目前在培养液中51班的数量是衡量每分钟计数从平均2分钟阅读。 </li><li>每分钟计数（CPM）读数的样品和对照使用，使用下列公式计算的具体裂解％: [（实验的CPM - 平均自发的CPM）/（平均最高CPM - 平均自发的CPM）] × 100 </li></ol> 8。代表性的成果: <img src="/files/ftp_upload/2668/2668fig1.jpg" alt="图1" /> 图1。从外周血自然杀伤细胞的隔离涉及的步骤和代HIV - 1感染的靶细胞。 <img src="/files/ftp_upload/2668/2668fig2.jpg" alt="图2" /> 图2。建设一个CD107a利用效应和K562细胞，未感染的CD4 + T细胞为目标的HIV - 1感染的T细胞NK细胞的脱颗粒检测中涉及的步骤。 <img src="/files/ftp_upload/2668/2668fig3.jpg" alt="图3" /> 图3。流式细胞仪门控 （一） 战略的一个CD107a脱粒检测单个细胞和不包括地积团块或双峰浇注森林管理委员会（前向散射面积）与FSC - H（前向散射高度）。 （二）绘制单细胞门FSC - A对SSC（侧散射）对淋巴细胞人口浇注。 （三）作为CD3/14/20绘制淋巴细胞门（T细胞，单核细胞，B细胞）和CD56（NK细胞）CD56的POS andCD3/14/20 NEG人口（NK细胞门）的门控。 （四）NK细胞门绘制CD107a VS CD56的可视化，有脱颗粒（CD107a POS）的NK细胞。 <img src="/files/ftp_upload/2668/2668fig4.jpg" alt="图4" /> 图4。在建设51铬释放利用效应和K562细胞，未感染的CD4 + T细胞为目标的HIV - 1感染的T细胞NK细胞的实验涉及的步骤。 <img src="/files/ftp_upload/2668/2668fig5.jpg" alt="图5" /> 图5。评估针对的自然杀伤细胞毒性响应代表结果艾滋病毒1感染的细胞。（一）NK细胞分别到degranulate没有目标和K562细胞的能力进行评估，首要的CD4 + T -细胞及艾滋病毒感染的主要T -细胞作为评估的NK细胞表面表达CD107a的百分比。在每个象限的数字代表总NK细胞的百分比。二）NK细胞是评估自己的能力在不同的效应细胞裂解K562细胞，未感染的CD4 + T细胞和HIV - 1感染的T细胞在 51铬释放法检测靶细胞比（E:T） 。

<ol>
	<li>Ruscetti, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+effector+mechanisms+against+HTLV-I-+and+HTLV-III/LAV-infected+lymphoid+cells.">Analysis of effector mechanisms against HTLV-I- and HTLV-III/LAV-infected lymphoid cells.</a> J Immunol. 136, 3619-3624 (1986).</li><li>Zheng, Z. Y., Zucker-Franklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+ineffectiveness+of+natural+killer+cells+vis-a-vis+retrovirus-infected+targets.">Apparent ineffectiveness of natural killer cells vis-a-vis retrovirus-infected targets.</a> J Immunol. 148, 3679-3685 (1992).</li><li>Ferrari, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clade+B-based+HIV-1+vaccines+elicit+cross-clade+cytotoxic+T+lymphocyte+reactivities+in+uninfected+volunteers.">Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers.</a> Proc Natl Acad Sci U S A. 94, 1396-1401 (1997).</li><li>Shankar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+function+of+circulating+HIV-specific+CD8(+)+T+cells+in+chronic+human+immunodeficiency+virus+infection.">Impaired function of circulating HIV-specific CD8(+) T cells in chronic human immunodeficiency virus infection.</a> Blood. 96, 3094-3101 (2000).</li><li>Ward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Vpr+triggers+natural+killer+cell-mediated+lysis+of+infected+cells+through+activation+of+the+ATR-mediated+DNA+damage+response.">HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.</a> PLoS Pathog. 5, e1000613-e1000613 (2009).</li><li>Ward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+modulates+the+expression+of+ligands+important+in+triggering+natural+killer+cell+cytotoxic+responses+on+infected+primary+T-cell+blasts.">HIV modulates the expression of ligands important in triggering natural killer cell cytotoxic responses on infected primary T-cell blasts.</a> Blood. 110, 1207-1214 (2007).</li><li>Bonaparte, M. I., Barker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+human+immunodeficiency+virus-infected+primary+T-cell+blasts+by+autologous+natural+killer+cells+is+dependent+on+the+ability+of+the+virus+to+alter+the+expression+of+major+histocompatibility+complex+class+I+molecules.">Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules.</a> Blood. 104, 2087-2094 (2004).</li><li>Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+mechanisms+in+cell-mediated+cytotoxicity.">Secretory mechanisms in cell-mediated cytotoxicity.</a> Annu Rev Cell Dev Biol. 23, 495-517 (2007).</li><li>O'Doherty, U., Swiggard, W. J., Malim, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+spinoculation+enhances+infection+through+virus+binding.">Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.</a> J Virol. 74, 10074-10080 (2000).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. 294, 15-22 (2004).</li><li>Fogli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysis+of+endogenously+infected+CD4++T+cell+blasts+by+rIL-2+activated+autologous+natural+killer+cells+from+HIV-infected+viremic+individuals.">Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.</a> PLoS Pathog. 4, e1000101-e1000101 (2008).</li><li>Tomescu, C., Chehimi, J., Maino, V. C., Montaner, 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=NK+cell+lysis+of+HIV-1-infected+autologous+CD4+primary+T+cells:+requirement+for+IFN-mediated+NK+activation+by+plasmacytoid+dendritic+cells.">NK cell lysis of HIV-1-infected autologous CD4 primary T cells: requirement for IFN-mediated NK activation by plasmacytoid dendritic cells.</a> J Immunol. 179, 2097-2104 (2007).</li><li>Ward, J. P., Bonaparte, M. I., Barker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-C+and+HLA-E+reduce+antibody-dependent+natural+killer+cell-mediated+cytotoxicity+of+HIV-infected+primary+T+cell+blasts.">HLA-C and HLA-E reduce antibody-dependent natural killer cell-mediated cytotoxicity of HIV-infected primary T cell blasts.</a> Aids. 18, 1769-1779 (2004).</li></ol>

正确完成后，在本协议中所述的检测应提供的NK细胞的能力degranulate对和裂解HIV - 1感染的细胞（见图5）的代表图片。在艾滋病毒感染的细胞和NK细胞的艾滋病毒感染的细胞裂解NK细胞脱颗粒应该是成正比10。两个NK细胞功能分析来衡量HIV感染的细胞的细胞毒性反应的可靠的结果是依赖于高度纯化的NK细胞以及高度纯化的人口受感染的细胞的隔离。经纯化的NK细胞和HIV - 1感染细胞的关键是实现一个相当准确的效应靶细胞比。同样，去除死亡和细胞凋亡的目标细胞群是重要的前51铬标签或与效应细胞的培育，51班正在经历死亡或凋亡细胞的存在，在同位素标记的步骤凋亡的细胞可能是由内在将导致高的自发释放，并会扭曲％计算具体裂解。此外，死亡或凋亡细胞的存在可能引发NK细胞脱颗粒，导致CD107a表达异常高的水平。精确移液是必要的，取出时无细胞上清液中NK细胞和靶细胞孵育 51铬释放试验，从每个重复删除上清量的差异会导致在高标准的偏差。可以修改这些协议，以评估具体的NK细胞受体的作用，在事先与对立抗体的特定受体或配体6,11共培养孵化的效应或目标触发NK细胞的艾滋病毒感染的细胞裂解。细胞因子治疗（如IL - 2，IL - 15）的NK细胞可用于评估刺激的12个NK细胞的功能。同样，抗体依赖性细胞毒性试验，可能会使用这些协议的执行。抗- HIV抗体（如抗- gp120的），可以增加识别的靶细胞，NK细胞低亲和力Fc 受体 CD16 13。这些检测，虽然成立来衡量HIV感染的细胞NK细胞的细胞毒性反应，也可以被修改来衡量的NK细胞的艾滋病毒感染的细胞识别后产生细胞因子的能力。虽然我们在本议定书中所描述的使用在体外感染的细胞作为靶细胞，我们最近所描述的靶细胞的感染艾滋病毒的患者代。这需要隔离的CD4 +细胞扩张过了两个星期的期间，在白细胞介素-2 11的存在有丝分裂原刺激T细胞。扩张后的两个星期内，在这篇文章中所述的协议，用于隔离的靶细胞。

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Vaccutainer Tubes (Sodium Heparin)</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>367874</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep CD4+ T-cell Isolation Kit</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>15062</td>
<td>&nbsp;</td>
<tr>
<td>CMF PBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30256.01</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocyte Separation Medium</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>25-072-CV</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-1640</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30096.01 </td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>25-005-Cl</td>
<td>&nbsp;</td>
<tr>
<td>T-cell Expansion Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-091-441</td>
<td>&nbsp;</td>
<tr>
<td>CMF HBSS (1x)</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30588.01</td>
<td>&nbsp;</td>
<tr>
<td>0.5M EDTA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>46-034-Cl</td>
<td>&nbsp;</td>
<tr>
<td>NK Cell Negative Isolation Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-092-657</td>
<td>&nbsp;</td>
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12440-046</td>
<td>&nbsp;</td>
<tr>
<td>CD4+ Positive Isolation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>113.31D</td>
<td>&nbsp;</td>
<tr>
<td>Dead Cell Removal Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>Polybrene</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-134220</td>
<td>&nbsp;</td>
<tr>
<td>CD3 PacificBlue</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>558117</td>
<td>&nbsp;</td>
<tr>
<td>CD14 PacificBlue</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>325616</td>
<td>&nbsp;</td>
<tr>
<td>CD20 PacificBlue</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>302328</td>
<td>&nbsp;</td>
<tr>
<td>CD56 APC</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>318310</td>
<td>&nbsp;</td>
<tr>
<td>CD69 PE</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>555531</td>
<td>&nbsp;</td>
<tr>
<td>CD107a FITC</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>555800</td>
<td>&nbsp;</td>
<tr>
<td>51Chromium</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEZ030002MC</td>
<td>&nbsp;</td>
<tr>
<td>Gamma Counter Tubes</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>1270-401</td>
<td>&nbsp;</td>
<tr>
<td>2470 Automatic Gamma Counter</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>2470-0050</td>
<td>&nbsp;</td>
<tr>
<td>FACS Diva</td>
<td>&nbsp;</td>
<td>Beckton Dickenson</td>
<td>643629</td>
<td>&nbsp;</td>
<tr>
<td>FlowJo</td>
<td>&nbsp;</td>
<td>TreeStar</td>
<td>FJ-9-1YR</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and use of hiv-1 infected primary cd4+ t-cells as target cells in natural killer cell cytotoxic assays

自然杀伤（NK）细胞病毒感染的细胞的先天免疫反应的一个重要组成部分。重要的是了解NK细胞识别并裂解HIV - 1感染细胞，因为任何aberrancy确定对HIV感染的细胞，NK细胞功能，可能会导致治疗，会提高其杀伤活性的能力。有必要使用的靶细胞，而再感染的T细胞系的细胞毒性检测艾滋病毒感染的主要T细胞爆炸。 T细胞系，即使没有感染，很容易受到NK细胞裂解。此外，它是必要的，使用自体的主要细胞，以防止主要组织相容性复合体类之间的目标和效应细胞，这将导致在裂解我错配。早期研究评估原发性HIV感染的细胞NK细胞的杀伤反应没有表现出明显的杀伤感染细胞 1,2 。然而，艾滋病毒感染的主要作为在NK细胞功能分析的靶细胞的T细胞已困难的，因为污染未受感染的细胞的存在。捐助者NK细胞功能的变化可能是由于样品之间的NK细胞杀伤的变化将导致这种不一致的感染细胞未受感染的细胞比例。因此，将有利于感染的细胞人口与纯化的工作，以标准化的效应为目标的实验 3,4之间的细胞比例。在这里，我们展示了HIV - 1病毒的能力优势，在受感染的细胞和商业套件的可用性向下调节CD4 +除去死亡或濒临死亡的细胞3-6高度纯化的HIV - 1感染细胞的人口隔离。纯化后的感染主要的T细胞可以被用来作为爆炸目标，在任何一个脱粒或效应人口5-7用纯化NK细胞的细胞毒性检测。使用在脱粒检测NK细胞效应评估NK细胞的能力， 释放 8所谓的"杀伤冲剂"的专门溶酶体裂解内容。通过与CD107a，溶酶体膜蛋白，成为NK细胞表面杀伤颗粒融合到质膜表达对一个荧光标记的抗体染色，我们可以判断在反应靶细胞识别degranulate NK细胞的百分比。另外，NK细胞的催化活性，可评估细胞毒性实验， 允许 51铬释放从内NK细胞的存在的靶细胞裂解的靶细胞比例的决心。

Watch this Scientific Journal Video about Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays at JoVE.com

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

细胞毒性实验来衡量HIV感染的细胞自然杀伤细胞裂解反应是有限的靶细胞的纯度。我们这里展示的高度纯化的HIV - 1感染的主要T细胞爆炸的人口利用HIV - 1病毒的能力，向下调整的CD4隔离。

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

preparation-use-hiv-1-infected-primary-cd4-t-cells-as-target-cells

Research

JoVE Journal

Immunology and Infection

25.9K 次观看.  Rush University Medical Center. 细胞毒性实验来衡量HIV感染的细胞自然杀伤细胞裂解反应是有限的靶细胞的纯度。我们这里展示的高度纯化的HIV - 1感染的主要T细胞爆炸的人口利用HIV - 1病毒的能力，向下调整的CD4隔离。

视频: Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

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.