Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

这项研究是由教育部，新加坡教育部（ACRF Tier1-RG40 / 13层2-MOE2013-T2-2-038）的支持。这份手稿是由来自Obrizus通信艾米沙利文编辑。

所有小鼠饲养和无特定病原体的条件下维持和所有小鼠协议按照机构动物护理和使用委员会的指导方针进行。 1.缓冲液和试剂的制备<ol><li>制备完整罗斯韦尔园区纪念研究所（RPMI）培养基（10％热灭活胎牛血清（FBS），2mM的L-谷氨酰胺，青霉素（100国际单位/毫升）/链霉素（100微克/毫升），55μM2-巯基乙醇）。 </li><li>准备20X平衡盐溶液（BSS）股票1和库存2，分别。 <ol><li>准备20X BSS股票1（111毫米葡萄糖，8.8毫米磷酸钾，26.7磷酸钠1升无菌水二元）和最终音量调整前加40毫升中含0.5％酚红20倍BSS股票1。无菌过滤器采用0.2微米的过滤器，在4℃下储存之前。 </li><li>制备20倍的BSS库存2（25.8毫氯化钙二水合物，107毫氯化钾，2.73酸钠沟道loride，19.6毫氯化镁六水合物，于1升无菌水16.6毫硫酸镁）。无菌过滤器采用0.2微米的过滤器，在4℃下储存之前。 </li><li>要准备1X BSS的实验使用，稀释50毫升BSS股票1和50毫升BSS股票2，另外，400毫升无菌水各。结合这两个稀释溶液，调整至pH 7，并添加20毫升FBS（2％）。顶起来使用无菌水和无菌过滤器使用0.2微米的过滤器1升。 注:BSS储液应单独准备，因为集中的BSS股票的混合，直接会导致沉淀。 </li></ol></li><li>红细胞（RBC）裂解液<ol><li>为了制备RBC裂解缓冲液，混合9份氯乙烯库存铵（155毫氯化物在无菌水中铵）在使用前1份股票Tris碱（在无菌水中130毫摩尔Tris（羟甲基）氨基甲烷，pH值7.65）。 注意:在4℃的氯化铵和Tris碱商店无菌原液。准备裂解buffer新鲜确保红细胞裂解效率。 </li></ol></li></ol> 2.代脾或淋巴结淋巴细胞悬液的注:以制备用于小鼠安乐死之前实验所需的所有试剂和设备，并尽快产生淋巴细胞的单细胞悬浮液，以维持高的细胞存活率是重要的。 <ol><li>颈椎脱位或CO 2窒息安乐死实验老鼠。 注意:这一步起，所有实验程序应无菌条件下进行。 </li><li>做任何切口之前浸整个鼠标放到70％的乙醇。取出脾和淋巴结无菌8，并将它们置于含5毫升的冰冷RPMI / FBS（RPMI用2％FBS）或BSS / FBS（从步骤1.2.3）独立15ml试管。 注:由于BSS防止高效RBC裂解，使用RPMI的脾细胞悬浮液的制备，并切换到BSS AFTE- [R RBC裂解。 </li><li>以产生从脾脏或淋巴结单细胞悬浮液，将器官（多个）在无菌100微米的细胞滤网目的两片在含有2毫升冰冷的RPMI / FBS或BSS / FBS的培养皿。使用1毫升注射器的柱塞，捣烂器官（S），直到它已被撕裂成很细的部分。 </li><li>细胞悬液转移到15毫升管中并洗涤细胞过滤用冰冷的RPMI / FBS或BSS / FBS的啮合。收集剩余的细胞悬浮液，将其添加到相同的15毫升管中，并在4℃下降速在453×g离心5分钟。去除上清。 注:加入RBC裂解缓冲液或培养基中的后续步骤之前轻弹用手指管重悬浮沉淀的细胞。 </li><li>将细胞悬浮液的离心分离过程中制备室温（RT）RBC裂解缓冲液（见步骤1.3）。制粒细胞并除去上清液后，再停止与每10 8个细胞1毫升RBC裂解缓冲液中的细胞。 INCU软化在RT裂解反应3-4分钟。 </li><li>停止RBC裂解14毫升冰冷的BSS / FBS和在4℃降速在453×g离心5分钟。 </li></ol> 3. B和T细胞的分离纯化<ol><li> B细胞的纯化<ol><li>算使用血球细胞。重悬在300微升BSS / FBS高达10 8脾细胞，并加入50微升抗CD43磁珠9,10。要去除死细胞，加入30微升Annexin V的磁珠。孵育在4℃冰箱内的细胞悬浮液30分钟。 </li></ol></li><li>性T细胞的分离纯化<ol><li>算使用血球细胞。重悬至10 8个细胞的非T细胞耗竭抗体混合物（抗CD19，B220，GR-1的生物素化抗体，TCR-γδ，CD49b，CD11c的，细胞CD11b，TER119和CD4或CD8根据目标细胞群体在200μlBSS / FBS -4,5- 200:被纯化），稀释。孵育在所述细胞悬浮液4℃冰箱中15分钟。 </li><li>孵育后，加入10 mL的BSS / FBS洗涤细胞，并在4℃下降速在453×g离心5分钟。去除上清，再暂停165微升BSS / FBS细胞与30微升链霉亲和微珠15微升Annexin V的磁珠。孵育在4℃冰箱内的细胞悬浮液30分钟。 注:为了确保即使在磁微珠标记，用微珠孵育细胞15分钟，然后通过轻敲15毫升管轻轻混合细胞悬液和步骤3.1.1期间孵育另外15分钟。 3.2.2或。 </li></ol></li><li>分离柱的制备细胞纯化<ol><li>细胞的微珠标记中制备的未使用分离柱（步骤3.1.1。或3.2.2。）。预暖的BSS（无FBS）至RT，并用2毫升给洗并无菌平衡色谱柱。用BSS平衡后，将洗涤塔不应该被允许干燥。 注:我们使用LSÇolumn代替推荐的LD柱的，由于其重复使用性（参见步骤3.4）。 </li><li>用磁珠标记后，添加14毫升BSS / FBS洗涤细胞，并在4℃下降速在453×g离心5分钟。除去上清液并重新悬浮在1-3毫升RT BSS细胞。 </li><li>附加的无菌21g的针列的前端纯化的过程中，以降低流速。加载细胞悬液到平衡柱，并通过含有纯化靶细胞收集的流动。 注意:避免引入气泡进入而载入列。 </li><li>用1毫升BSS / FBS冲洗柱子一次，并通过包含纯化的靶细胞采集流程。通过再次刷新同流列。通过在相同的15ml试管第二装载后收集的流动。 </li><li>用1ml BSS / FBS洗涤柱3次，并通过含有纯化靶细胞收集的流动。此后，加5毫升BSS / FBS为thÈ柱，有柱塞，冲洗磁性标记的细胞出列到一个新的15ml试管中。 </li><li>检查由流收集仪使用结合到表面纯化的B细胞或T细胞4,5抗原的抗体的细胞的纯度。 </li></ol></li><li>重新使用分离柱 注:LS柱可以在不影响净化效率被重用高达4倍。 <ol><li>用5ml磷酸盐缓冲盐水（PBS）中，用从使用柱塞顶端蒸馏水洗5×3次洗涤柱3次。 </li><li>洗用5毫升70％的乙醇柱和广泛使用空气抽头防止生锈的该列中的积聚干燥塔。 </li><li>以制备用于单独的纯化实验中使用的LS柱，从下向上洗柱，使用注射器接合件5 1ml 70％乙醇。接着，用5毫升无菌PBS，从塔的顶部，随后用5毫升的PBS洗一次从底部向上的柱两次。添加2RT毫升BSS平衡柱，然后进行与标记细胞装入列。 </li></ol></li></ol> 4. CFSE标记和刺激注:纯化的细胞可以经受多种在体外和体内功能性测定法。在这里，我们使用纯净的T细胞，以确定装甲运兵车5的T细胞的刺激能力。 <ol><li>预暖标签解决方案（PBS中0.1％FBS）至37°CFSEÇ之前加载。 注意:使用的FBS的PBS中的低百分比的CFSE装载期间减少细胞死亡和在离心分离期间最大限度地减少细胞的损失。然而，过多的FBS可以用CFSE载荷干扰。 </li><li>用标记溶液洗涤纯化的细胞两次，然后在15毫升管预热的标记溶液再悬浮为2×10 7个细胞/ ml。 </li><li>制备10μM的CFSE溶液（1:500稀释的5mM CFSE原液）的预热的标记溶液。 CFSE解决方案应每次实现最佳标记新鲜配制。 </li><li>加载细胞用CFSE，在37℃下添加1份细胞悬液至1份10微米的CFSE溶液在一个15毫升管中并孵育在黑暗中10分钟。为5μMCFSE的最终浓度用于标记1×10 7个细胞/ ml。 </li><li>倒置该管每隔2分钟，以确保CFSE装载期间细胞的均匀的混合物。 </li><li>停止反应，加入冰冷的完全RPMI介质的多个卷，且在4℃降速在453 XG 5分钟。在成功CFSE加载，将细胞沉淀将出现淡黄色。 </li><li>洗涤CFSE装载的细胞再一次用冰冷的完全RPMI培养基中，并使用用于体外培养或体内刺激前在4℃降速在453×g离心5分钟。 </li></ol> 5. 体外刺激<ol><li>临用前准备刺激的2倍原液（2X刺激原液），使100微升2×刺激原液可以加入到100μl的细胞对每孔200微升的在一个96孔板的最终体积。 </li><li>如果需要涂覆有刺激（IgM抗体或CD40对B细胞或CD3和CD28对于T细胞）的板，稀释在PBS中的刺激和预涂层的培养板在4℃下过夜。可替代地，将培养板可在37℃被涂布于在实验当天1小时。用PBS（不要让盘在任何时间干）洗涂板的两倍。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 对于B细胞 </td><td></td></tr><tr><td> 刺激 </td><td> 最终浓度 </td></tr><tr><td>的F（ab&#39;）2山羊抗小鼠IgM </td><td> 0.6-2.4微克/毫升</td></tr><tr><td>抗小鼠CD40单克隆抗体</td><td> 0.5-2微克/毫升</td></tr><tr><td>重组小鼠IL-4 </td><td> 25单位/毫升</td></tr><tr><td>脂多糖</td><td rowspan="2"> 0.1-10微克/毫升</td></tr><tr><td> （LPS）的来自大肠杆菌大肠杆菌血清型055:B5 </td></tr><tr><td></td><td></td></tr><tr><td> 对于T细胞 </td><td></td></tr><tr><td> 刺激 </td><td> 最终浓度 </td></tr><tr><td>抗CD3（板涂层） </td><td> 2-10微克/毫升</td></tr><tr><td> （50μl/孔涂覆） </td><td></td></tr><tr><td>抗CD28（板涂层） </td><td> 2微克/毫升</td></tr><tr><td>重组IL-2 </td><td> 40单位/毫升</td></tr><tr><td> PDBu组（佛波酯） </td><td> 5-50纳克/毫升</td></tr><tr><td> A23187（钙离子载体） </td><td> 250纳克/毫升</td></tr></tbody></table> 表1:用于刺激在体外培养的淋巴细胞的刺激的浓度。<ol start="3"><li>对于CFSE标记的B细胞，重新悬浮在一式三份的完全RPMI培养基和培养至3×10 6个细胞/ ml用3×10 5细胞中/孔72小时的96孔平底板。 </li><li>对于CFSE标记的T细胞，再悬浮于完全RPMI培养基和培养，一式三份用0.5-3×10 5个细胞0.5-3×10 6个细胞/ ml /孔在96孔48或72小时圆底平板。 </li></ol> 6. 在体刺激<ol><li> 用于体内刺激，过继转移4×10 6每只小鼠CFSE标记的T细胞（静脉内（IV）在200μlPBS中）到每个MHC匹配的受体小鼠。 注:在这个协议中，CFSE标记的T细胞可以使用继尾静脉或眼眶注射，因为这些细胞将家里淋巴器官如脾和淋巴结转移。 </li><li>挑战受体小鼠一天后的抗原。 注意:在这个例子中，使用卵白蛋白（OVA蛋白，50微克/小鼠）作为抗原，因为OVA特异性T细胞受体（TCR）-transgenic T细胞过继转移到受者小鼠。准备OVA蛋白无菌PBS并注入100微升OVA蛋白/ PBS或PBS控制，通过皮下注射（SI），为每个收件人鼠标5。 </li><li>收获并用OVA蛋白或PBS免疫接种后3天生成从淋巴器官受体小鼠的单细胞悬浮液（淋巴结和脾）。单独的淋巴结到近端淋巴结（PLN），其中包括腋窝，臂和颈浅淋巴结）和远端淋巴结（DLN），它包括肠系膜，腘，腹股沟，腰椎，和尾部淋巴结。使用适当的FACS抗体染色的细胞以检查T细胞增殖。 注:在此CFSE细胞跟踪实验，CFSE标记的T细胞PBS注射的对照小鼠建立一个基线荧光非二viding细胞。增殖的细胞分裂，抗原刺激，CFSE标记的细胞通过测量荧光峰12,13可视化。与PBS对照，增殖的细胞分裂的次数，CFSE标记的T细胞可以被确定12,13。 </li><li>通过比较细胞分裂或峰的样本之间的数字分析CFSE标记的细胞增殖数据（ 图1和2）。 注意:例如，CFSE标记，继转移到接收到的OVA抗原受体小鼠OVA特异性T细胞将较之PBS注射的对照小鼠5,12经历活跃增殖。此外，值得注意的是要指出，有很多方法来分析CFSE细胞增殖数据，由Hawkins和同事14所证明。 </li></ol>

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D., Hommel, M., Turner, M. L., Battye, F. L., Markham, J. F., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nat. Protoc. 2 (9), 2057-2067 (2007).</li><li>Tomlinson, M. J., Tomlinson, S., Yang, X. B., Kirkham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation:+Terminology+and+practical+considerations.">Cell separation: Terminology and practical considerations.</a> J. Tissue Eng. 4 (1), 1-14 (2013).</li></ol>

The authors have nothing to disclose.

在这个协议中，我们展示了从淋巴器官淋巴细胞净化的过程。使用磁珠分选的细胞纯化是产生可行的，高度纯化的靶细胞快速和简单的方法。 该议定书中的关键步骤 细胞活力和细胞产量 体外造血保持系细胞的活力，是确保成功的和可重复的实验，是至关重要的。化学和生物试剂，次优实验条件或切除器官的不适当...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

淋巴细胞的磁性细胞纯化允许用户在一段相对短的量纯化的靶细胞群。使用我们的耗尽协议中，我们能够从72.8％（前纯化）增加的CD8 T细胞的百分比（OT-I在重组活化基因-1（RAG-1）缺陷小鼠）到94.2％（纯化后; 图1A）4,5。然后将这些纯化的淋巴细胞可用于下游功能测定来确定的淋巴细胞增殖和信号转导4,5。例如，我们可以研究的装甲运兵车?...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

21.0K 次观看.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

视频: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

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 ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

癌症研究

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

鼠淋巴结基质细胞的分离

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

从小鼠真皮淋巴毛细血管中分离内皮细胞

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

作者聚焦：基于磁珠的小鼠真皮淋巴内皮细胞分离

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

小鼠真皮淋巴内皮细胞分离

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...