D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

在这项研究中，成年（6-8周）雌性C57BL / 6小鼠。小鼠根据根特大学动物饲养的指导原则安置和饲养。所有动物的程序获得批准的机构动物护理和伦理委员会。 1.准备单核从小鼠脾细胞（跨国公司）的<ol><li>通过颈脱位牺牲鼠标。 </li><li>把鼠标放在解剖板背下来，并修复后和前肢与引脚。 </li><li>消毒皮肤表面，用70％（体积/体积）乙醇/ H 2 O </li><li>切割沿腹部中线到胸部皮肤和使用无菌手术器械暴露脾脏。 </li><li>修整周围脂肪组织后，将脾脏成24孔板含1ml 1×磷酸盐缓冲盐水（PBS）在室温下。 </li><li>用无菌手术刀解剖脾脏和部分转移到70微米的尼龙过滤器放置在50毫升的浴缸即</li><li>采用2毫升注射器活塞，轻轻捣烂脾片通过细胞过滤和洗涤用5毫升1X PBS。 </li><li>加入3 ml的菲可帕克的至15ml管中并覆盖与5 ml的细胞悬浮液的密度梯度介质。 </li><li>离心在400×g离心20分钟，在RT下无制动。 </li><li>相间转移到新鲜15ml试管，加入10毫升冷的1×PBS和离心机在300×g离心10分钟，在4℃。 </li><li>重悬在2ml的1×PBS含有0.5％牛血清白蛋白（BSA）和1mM乙二胺四乙酸（EDTA）（FACS缓冲液）中的细胞。转移10微升细胞悬浮液到一个小管，混合，用10微升0.4％的</li></ol> 2.富集，检测和鼠标iNKT细胞的分离纯化<ol><li>富集的小鼠脾脏CD5positive（CD5 +）淋巴细胞。 <ol><li> （可选的）染色10 5个细胞与FITC缀合的抗CD5（4微升1点半稀释液/ 10 6个细胞）30分钟，在4℃下在黑暗中，洗净用200μlFACS缓冲液重悬在500μlFACS缓冲液。准备的4&#39;，6-二脒基-2-苯基吲哚（DAPI）在1×PBS中20μM的工作溶液中，加入1微升其至10 5个细胞在FACS缓冲液，并在室温下孵育5分钟。获得10 4个预富集细胞上的流式细胞仪。 <ol><li>使用FCS-H和FCS-W，电栅极上的FSC-W 罗细胞以排除细胞双峰和聚集物（ 图1A）。然后，电子门出DAPI +（死）了FSC-W 罗群（ 图1B）内的细胞，并使用SSC-A和FCS-A按大小（ 图1C）电子选择淋巴细胞群。 </li><li>使用淋巴细胞门控内的细胞，画出SSC-A与CD5和如图1D电子栅极CD5 +淋巴细胞。获取的流式细胞仪的预富集的样品的剩余部分。 </l我&gt; </ol></li><li>调整细胞计数，以每90微升10 7个细胞在FACS缓冲液，并添加10微升抗CD5微珠。 </li><li>孵育4ºC15分钟，用4ml FACS缓冲液重悬在500μl冷FACS缓冲液洗一次。富集CD5 +使用磁性细胞分离（MACS）淋巴细胞根据制造商的建议列。 </li><li>可选）染色10 5个细胞与FITC缀合的抗CD5（4微升1:30稀释/ 10 6个细胞）和DAPI作为2.1.1。收购10 4富集淋巴细胞的流式细胞仪，并验证电子门2.1.1创建的。选择CD5 +淋巴细胞中富集的细胞部分。获得10 5个细胞的流式细胞仪分析的比例存在于富集馏分CD5 +淋巴细胞如图1E - 小时。 </li></ol></li><li>检测和小鼠的iNKT CE隔离LLS用流式细胞仪。 <ol><li>重悬富集CD5 +淋巴细胞在约10 6个细胞每20微升在含有抗CD16 / CD32的FACS缓冲液（4微升1:50稀释/ 10 6个细胞）和PE缀合的αGalCer的浓度/ CD1d的四聚体（1微克/ 10 6个细胞）。 </li><li>下室温温育30分钟，在黑暗中。 </li><li>稀释小鼠抗体（mAbs）的抗CD3εV500（4微升1:20稀释/ 10 6个细胞），抗-CD19 E450（4微升1:50稀释/ 10 6个细胞）和抗CD8αE450（4微升1:50稀释/ 10 6个细胞）在FACS缓冲液中，添加到CD5 +淋巴细胞和在黑暗中温育30分钟，在4℃。 </li><li>通过在300×g离心FACS缓冲液离心10分钟，在4℃下重悬在4毫升，以10 7个细胞/ ml的终浓度清洗细胞用1ml的FACS缓冲液中。 </li><li>通过30微米的细胞滤网过滤细胞，然后将细胞在冰上。 </li><li>校准流式细胞仪对细胞根据制造商的建议14,15排序。 注意:流式细胞仪的细胞分选的校准需要培训，并且可以由有经验的操作者，或受过训练的技术人员进行。 </li><li>加入10微升的DAPI每10 6个细胞20μM的工作溶液，并在室温下孵育5分钟。获得大约10 4富集CD5 +淋巴细胞上的流式细胞仪。上FSC-W 罗细胞电子门，以排除细胞双峰和聚集物（ 图2A）。然后，电子门出DAPI + CD8 + CD19 +细胞 （转储通道）的FSC-W 罗群（ 图2B）内，并使用SSC-A和FCS-A作为2.1.1.1中描述的电子选择淋巴细胞（ 图2C） 。 <ol><li>剧情αGalCer/ CD1d的四聚体由CD3ε和电子门αGalCer/ CD1d的如图2D四聚体+CD3ε+ iNKT细胞。获得不超过2×10 5个CD5 +富集的淋巴细胞，以确定百分比αGalCer/ CD1d的四聚体+CD3ε+ iNKT细胞存在于富集细胞部分。 </li></ol></li><li>利用在2.2.7中创建的电子门。使用70微米的喷嘴处的&#39;1.5&#39;的流速，FACS排序αGalCer/ CD1d的四聚体+CD3ε+ iNKT细胞上的流式细胞仪，在70磅。收集在含有1ml 100％FCS一FACS管排序iNKT细胞。接着用10ml RMPI 1640培养基和离心将细胞在300×g离心10分钟，在4℃下冲洗从FACS管侧排序iNKT细胞。 </li><li>重悬排序iNKT细胞在1ml的RPMI 1640培养基中，并获得40微升其上的流式细胞仪评估排序iNKT细胞的纯度。选择FSC-W LO DAPI -淋巴细胞参考译文]在2.2.7所述。 （ 图2E - G）和电子栅极αGalCer/ 如图2H的CD1d四聚体+CD3ε+ iNKT细胞。 </li></ol></li></ol> 3.文化和扩展鼠标iNKT细胞 0天<ol><li>外套平底96孔板中，每孔纯化的抗CD3ε（3微克/毫升）处理1小时，在室温。用200μl的1×PBS和一次用200μl的完全RPMI 1640培养基（10％体积/体积的FCS，100U / ml青霉素 - 链霉素，5.5μMβ巯基乙醇）洗涤两次。 </li><li>计数使用0.4％血球作为可行排序iNKT细胞（步骤2.2.8）的数量在1.11，离心机在300×g离心10分钟，在4℃，重悬在5×10 5个细胞/在完全RPMI 1640培养基毫升含有重组鼠IL-2（10毫微克/毫升），重组鼠IL-12（1纳克/毫升）和可溶性抗小鼠CD28（5微克/毫升）。薄板S在抗CD3ε涂层钢板orted iNKT细胞在10 5个细胞/孔，并在 CO 2培养箱中在37℃下进行2天。 </li></ol>第2天<ol start="3"><li> 在 CO 2培养箱转移细胞到一个新的未涂覆的平底96孔板中并培养静止的条件下将细胞在完全RPMI 1640培养基中，在37℃下进行2天。 </li></ol>第4天<ol start="4"><li>通过温和移液移走从培养板上的细胞，将细胞转移到一个新的15ml试管。离心机在300×g离心10分钟，在4℃下，悬浮在5×10 5个细胞/在完全RPMI 1640培养基毫升含重组鼠IL-7（5毫微克/毫升），和板10 5细胞/孔在将平在 CO 2培养箱底的96孔板4天，在37℃。 </li></ol> 8天<ol start="5"><li>收集细胞的新管，离心300×g于4℃下用含有可溶性抗CD28（5微克/毫升）10分钟，并重新悬浮于5×10 5细胞/ ml，在完全RPMI 1640培养基。 </li><li>外套平底96孔板中，每孔纯化的抗CD3ε（3微克/毫升）洗如3.1。板10 5个细胞/孔抗CD3ε包被的板并在37℃孵育在 CO 2培养箱直到11天。 </li></ol>第11天 - 18 <ol start="7"><li>重复步骤3.4至3.6。 </li></ol> 19-20日<ol start="8"><li>转移细胞到一个新的15ml试管中，离心以300×g离心，重悬在5×10 5细胞/ ml，在完全RPMI 1640培养基。 </li><li>板的细胞在10 5个细胞/孔在平底96孔板中，并允许细胞休息2天，在37℃在一个实验使用之前CO 2培养箱。 </li></ol> 4，细胞因子生产用鼠标iNKT细胞使用的CD1d开发平台电子商务结合的分析<ol><li>外套平底96孔板用100μl重组鼠的CD1d的（在1×PBS中10微克/ ml）的每孔，孵育1小时，在37℃。 </li><li>用200μl的1×PBS洗涤四次，在1×PBS（封闭溶液），每孔加入200微升2％FCS的孵育2小时，在37℃。 </li><li>除去封闭溶液，每孔添加200微升αGalCer（5毫微克/微升），孵育所述板在37℃下16小时。 [对于制备αGalCer溶液，加1085μ1×PBS中至55微克αGalCer的溶于含96毫克/毫升蔗糖，10毫克/毫升的脱氧胆酸钠和5毫克/毫升吐温20的车辆。 </li><li>温育后，弃去αGalCer溶液和洗两次以及用200μl1×PBS中，随后通过加入200μl的完全RPMI 1640培养基。 </li><li>收集在体外扩增的 iNKT细胞在第21天，离心将细胞在300×g离心10分钟，在4℃下重悬在完成RPMI 1640培养基以0.5×10 6细胞/ ml的浓度。 </li><li>加入200μl每孔的细胞的iNKT再悬浮并在 CO 2培养箱中孵育72小时，在37℃。 </li><li>通过根据制造商的协议酶联免疫吸附试验（ELISA）收集感兴趣的上清液并测量细胞因子。 </li></ol>

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The authors have no competing interests to disclose.

在当前协议关键步骤包括分离和CD5 +淋巴细胞（第1和2）的随后的富集，FACS分选（第3节）和iNKT细胞的初始接种（第4节）。第1节执行的步骤，切记要仔细层中的脾细胞悬浮液密度梯度介质，使得不同的细胞相间的离心后产生的。随后的富集CD5 +淋巴细胞通过磁性细胞分离（第2部分）最大程度地减少染色所需的试剂和时间和FACS分类iNKT细胞（第3节），这有助于增加的iNKT细胞产量后的排序。最后，种子每孔不超过10 5 iNKT细胞的第2天的培养（第4节）作为接种井具有更高数目的iNKT细胞可导致细胞凋亡是重要的。 修改和/或troubleshoo时，许多因素都是重要的婷这个协议:首先，所用动物的年龄是非常重要的。动物是太旧，或有附件的感染，可能会阻碍你的能力来分离，区分和流式细胞仪排序足够的细胞从脾。其次，与αGalCer/的CD1d四聚体染色时，它保持细胞悬浮于PBS的体积缓冲液低过高的体积稀释了αGalCer/ CD1d的四聚体，这又减小四聚体的聚类的染色效率是重要+ iNKT细胞用流式细胞仪。理想情况下，紧密集群αGalCer/ CD1d的四聚体+ iNKT细胞的流式细胞仪采集过程中存在允许更精确的门用流式细胞仪，并在后期的排序分析产生更高的百分比纯度iNKTs。这是重要的，因为低排序的iNKT细胞产量会导致其它污染的T细胞群的培养过程中的产物。第三，不断刷新介质的iNKT成为行政长官的文化和膨胀的过程中黄LLS。例如，特别是在天8和培养了11天更换泛黄介质与含有适当的细胞因子，新介质的50％。的iNKT细胞产量可以被最大化以这种方式。 据我们所知，这是第一次的iNKT膨胀协议为包括密度梯度和CD5 +淋巴细胞的富集步骤，用于分离和iNKT细胞的富集从小鼠脾脏。一些替代方法已探索以富集脾iNKT细胞包括αGalCer/ CD1d分子二聚体13，Vα14TCR Tg小鼠10以及非T细胞的耗竭磁性细胞分离9。然而，尽管使用这些方法iNKT细胞可成功地在体外扩增，这样的方法需要或大体积的αGalCer/ CD1d的二聚体以富集iNKTs 13中，仅生成转基因iNKT细胞10，或同时产生一些非的iNKT细胞系9 &lt;/ SUP&gt;。此外，使用板结合的抗CD3ε的维持和扩大有序iNKT细胞（第4节）期间的iNKT 体外扩增9排除为激活装甲运兵车的要求。在这种情况下，一个APC -自由的iNKT膨胀协议具有不必的APCs从膨胀iNKT细胞在注射前在体内分离的优点。我们应该但是也可以补充一点，描述的方案是由研究者需要容易获得，以及相关的训练，来校准和操作在FACS分拣机的事实的限制。不幸的是FACS分拣机是昂贵的，他们并不总是用在每一个实验室。然而，尽管目前的协议已被优化，以分离和培养的鼠iNKT细胞，也可以用于本文所述的富集步骤来分离和研究的iNKT胸腺近期移民（实时评价）16和可适于以富集和表征其他稀有Ť细胞群通过流式细胞仪。 8 iNKT细胞从三只小鼠的脾中3个星期内的优化方法。以这种方式产生的iNKT细胞保留刺激后分泌细胞因子的能力，使它们可用于过继转移实验的iNKT和细胞生物学的体内研究。

鼠不变的自然杀伤T（iNKT细胞）细胞是由CD1d的表达皮质胸腺细胞1,2在胸腺选择的先天T淋巴细胞的独特群体。 iNKT细胞表达由一个不变的Vα14-Jα18TCR链配对任Vβ8，Vβ7或Vβ2TCR的3，其能够识别内源性以及外国脂质抗原的CD1d的上下文中的T细胞受体（TCR）。例如，鼠iNKT细胞识别并通过内源性脂质抗原称为isoglobotrihexosylceramide（iGb3）4，以及α-半乳糖神经酰胺（αGalCer）5,6-，糖脂自海绵中分离被激活。 TCR依赖性iNKT细胞的活化促进适应性免疫应答的引发，并作为其结果，iNKT细胞已被证明是功能上参与的范围病状包括风湿性疾病7和癌症的改善或发展&lt;SUP&gt; 8。目前，合成的iNKT细胞的配体构成有希望的新的疫苗佐剂可以是能够调节的一些免疫病理状况。 先前已经证明，iNKT细胞可以在体外从以下然而小鼠组织分离来产生;许多研究中采用的使用主要抗原呈递细胞（APC）和/或细胞系9，Vα14TCR转基因（Tg）为10只小鼠，或胸腺瘤为的iNKT细胞衍生的杂交瘤11,12的产生。此外，大量的老鼠，大批量试剂如αGalCer加载CD1d的二聚体，和漫长的文化时代使一些出版协议不太道德和经济上的吸引力9,13。 在本报告中，我们描述了用于分离和在 iNKT细胞的小鼠脾脏体外扩增的方法适于。更具体地，协议描述了一种方法富集从小鼠脾脏这减少了所需的用于FACS细胞分选小鼠，试剂和时间，并提出了在体外扩展排序脾iNKT细胞的优化方法iNKT细胞。

<table><tbody><tr><td>Material</td><td></td><td></td><td></td></tr><tr><td>recombinant murine IL-2</td><td>eBiosciences</td><td>14-8021</td><td></td></tr><tr><td>recombinant murine IL-12</td><td>eBiosciences</td><td>39-8122-65</td><td></td></tr><tr><td>recombinant murine IL-7</td><td>eBiosciences</td><td>14-8071</td><td></td></tr><tr><td>purified anti-mouse CD3e (145-2C11)</td><td>eBiosciences</td><td>16-0032-86</td><td></td></tr><tr><td>purified anti-mouse CD28 (37.51)</td><td>eBiosciences</td><td>16-0281-85</td><td></td></tr><tr><td>e450-conjugated anti-mouse CD19 (eBio1D3)</td><td>eBiosciences</td><td>48-0193-82</td><td></td></tr><tr><td>e450-conjugated anti-mouse CD8&amp;alpha; (53-6.7)</td><td>eBiosciences</td><td>48-0081-82</td><td></td></tr><tr><td>FITC-conjugated anti-mouse CD5 (53-7.3)</td><td>eBiosciences</td><td>11-0051-81</td><td></td></tr><tr><td>V500-conjugated anti-mouse CD3&amp;epsilon; (500A2)</td><td>BD biosciences</td><td>560771</td><td></td></tr><tr><td>anti-mouse CD5 (Ly-1) microbeads</td><td>Macs Miltenyi Biotec</td><td>130-049-301</td><td></td></tr><tr><td>purified anti-mouse CD16/32 (2.4G2)</td><td>Macs Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>MACS MS Columns</td><td>Macs Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Trypan Blue, 0.4% (wt/vol)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Gibco</td><td>12633-020</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010-056</td><td>[Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; - free]</td></tr><tr><td>Fetal calf serum</td><td>Gibco</td><td>10270</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-123</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458-100ML</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906-100MG</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>EDS-1KG</td><td></td></tr><tr><td>Ficoll-Paque plus</td><td>GE Healthcare</td><td>71-7167-00 AF</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>96-well plate F-bottom</td><td>Greiner-bio one</td><td>657-160</td><td></td></tr><tr><td>24-well plate</td><td>Greiner-bio one</td><td>665-180</td><td></td></tr><tr><td>6-well plate</td><td>Greiner-bio one</td><td>655-180</td><td></td></tr><tr><td>15 ml falcon tube</td><td>Greiner-bio one</td><td>188-271</td><td></td></tr><tr><td>50 ml falcon tube</td><td>Greiner-bio one</td><td>227-261</td><td></td></tr><tr><td>70 &amp;micro;m filter</td><td>Greiner-bio one</td><td>542-070</td><td></td></tr><tr><td>30 &amp;micro;m filter</td><td>Millipore</td><td>SVGP01050</td><td></td></tr><tr><td>MiniMACS separator</td><td>Macs Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MS Columns</td><td>Macs Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Water-jacketed CO2 incubator</td><td>VWR</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>VWR</td><td></td><td></td></tr><tr><td>Dissection Kit</td><td>VWR</td><td></td><td></td></tr><tr><td>BD FACSAria III</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

刺激后迅速分泌细胞因子的能力是不变的天然杀伤T（的iNKT）细胞谱系的功能特性。因此iNKT细胞的特征是一种天生的T细胞群体能够激活和转向适应性免疫应答。的改进的技术用于小鼠的iNKT细胞的培养和扩大发展能促进的iNKT细胞生物学中的体外和体内模型系统研究。这里，我们描述了分离和扩增的鼠脾iNKT细胞的优化过程。 从C57BL / 6小鼠脾脏被除去，解剖和紧张，将所得细胞悬浮液被层叠在密度梯度介质。离心后，脾单核细胞（MNCs）被收集和CD5阳性（CD5 +）淋巴细胞富集使用磁珠。是在CD5 +分数在iNKT细胞随后用^5;神经酰胺加载的CD1d四聚体和通过荧光激活细胞分选（FACS）纯化。 FACS分选iNKT细胞然后最初在体外培养的小鼠重组IL-7存在下被扩展之前使用的重组鼠细胞因子和板结合的T细胞受体（TCR）的刺激的组合。使用这种技术，大约可以在18-20天的培养，之后可用于功能测定法在体外 ，或在小鼠体内转移实验，他们会产生10 8 iNKT细胞。

使用密度梯度脾的单核细胞的分离需要大约1小时，并消除了使用以裂解红血细胞（红细胞）所需的试剂的。使用这种方法和碎屑的脏器应变被去除期间产生得到一种高收率的活细胞。通常，iNKT细胞的脾淋巴细胞池内的频率总T淋巴细胞的1至5％的范围内然而，这可以根据所使用的动物的年龄，性别和健康状况而变化。大约10 6 iNKT细胞可以从3只小鼠的合并脾和产量最高iNKT细胞来获取从6-8周龄小鼠脾脏被获得。 使用小鼠抗CD5磁珠显著丰富的脾MNC级分中iNKT细胞，除此表达该CD5抗原的B淋巴细胞的未成年人口，多数细胞使用这一步骤中分离构成CD5 +淋巴核细胞。例如，CD5浓缩后获得的跨国公司5-8％的CD5阴性（ 图1H）。为的iNKT细胞的FACS分选，结合αGalCer/ CD1d的四聚体和抗小鼠CD3ε产生的iNKT细胞纯度在98％以上然而，流式细胞仪转储通道脾B和CD8 T细胞应用于消除排序期间"粘性"的淋巴细胞群。此外，栅极指出可能在CD5富集步骤形成的任何双峰。使用这种流式细胞仪的设置，我们在99％（ 图2H）的顺序获得的iNKT细胞纯度后排序。 在IL-2，IL-12和可溶性抗CD28的存在下刺激10 6分选iNKT细胞与板结合的抗CD3ε导致以下2天的培养（图3）的iNKT号码的3倍的扩张。随后的膨胀iNKT细胞中的IL-7的结果在3到4倍的增加存在于中培养4天iNKT细胞的数量的存在。 ñotably，重复这些培养条件可以产生平均的两轮膨胀后7×10 7 iNKT细胞没有细胞的变化对不同天培养介质之间的任何可见的损失（图3）。因此，鼠脾iNKT细胞可以扩展使用这些条件的18天为至少70倍。 我们还评估了扩大iNKT细胞的细胞因子产生能力通过刺激板结合的重组鼠的CD1d装入αGalCer。在48小时的刺激后，IL-2，IL-4，IFN-γ，TNF-α和IL-6的可容易地在培养物上清液中通过ELISA检测（图4）。因此，扩大的iNKT细胞保留产生Th1和Th2细胞因子后扩张的能力。 <img alt="图1" src="/files/ftp_upload/53256/53256fig1.jpg" /> 图1.丰富的CD5 +淋巴细胞从小鼠脾脏跨国公司 。门控策略进行分析CD5 +淋巴细胞中存在的预富集的（AD）和磁性细胞分离富集的（EH）小鼠脾的MNC悬浮液的百分比。细胞双峰（A，E）和死细胞（B，F）已排除使用FSC-H与FCS-W和DAPI与FSC-A地块，分别为。百分比CD5 +细胞淋巴细胞门（C，G）之前和富集内出现如图D和H分别。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53256/53256fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53256/53256fig2.jpg" /> 无花果<html> URE 2. FACS分拣从CD5富集脾跨国公司iNKT细胞。门控策略分析的百分比αGalCer/前存在的CD1d四聚体+CD3ε+ iNKT细胞（A - D）和之后（E - 高）FACS分类。门控策略消除细胞双峰（A，E），死细胞和从分析CD8 + CD19 +淋巴细胞（B，F）通过绘制FSC-H与FCS-W和DAPI，CD8和CD19（转储通道）与FSC- A，分别。百分比αGalCer/前和FACS分选后淋巴细胞门控（C，G）内本的CD1d四聚体+CD3ε+ iNKT细胞示于（D）和（H）分别。 CD1d的-春节，αGalCer/ CD1d的四聚体。获得="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53256/53256fig3.jpg" /> 体外产生的 iNKT细胞的图3的绝对数目 。倍的iNKT细胞在指定的时间点3周体外扩增后的数量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53256/53256fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53256/53256fig4.jpg" /> 图4.细胞因子在体外生产发泡iNKT细胞的能力。第20天扩大iNKT细胞刺激在体外用鼠αGalCer负载CD1d的。后，收集并用于我的存在下进行分析72小时的上清液FN-γ，IL-4，TNF-α，IL-2和IL-6的ELISA法。酒吧代表表示SEM（N = 2）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53256/53256fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

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

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 ...

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Nanyang Technological University

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Immunology and Infection

11.9K 次观看.  Ghent University Hospital. 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. 

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

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Isolating Invariant Natural Killer T Cells from a Mouse Model

Source: Meng, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60048">Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice.</a>&#160;J. Vis. Exp.&#160;(2020)
This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

从小鼠模型中分离不变的自然杀伤 T 细胞

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation

Source: Nguyen, H. D., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp. (2020).
This video describes T-cells enrichment from the spleen of a mouse by negative selection using a magnetic separator. The technique involves targeting the other cells in the sample for depletion using antibodies or ligands directed against specific cell surface antigens.

T 细胞富集:一种通过磁分离从混合细胞群中分离 T 细胞的技术

Source: Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. ...

癌症研究

白血病

Cell isolation & characterization

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Source: Buchele, V., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58871">Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

通过同种异体骨髓和脾细胞移植在小鼠模型中诱导移植物抗宿主病

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. GvHD ...

Immunopathology

Host-Pathogen Interaction Models

Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

To isolate antigen-specific CD8-positive T cells, macerate the spleens and lymph nodes from wild-type C57Black/6j mice through a sterile 70-micrometer cell strainer with frequent PBS rinses, and use a no-touch CD8-positive T cell isolation kit, according to the manufacturer&#39;s instructions, to eliminate the non-CD8-positive T cells.
After counting, collect the isolated CD8-positive T cells by centrifugation, and resuspend the pellet in 100 microliters of PBS, supplemented with 0.5% bovine serum albumin and 2-millimolar EDTA.
Then, incubate the cells with the nanoparticle artificial antigen-presenting cells such that there are 1 x 1011 peptide-loaded MHC-Ig, per 1 x 106 isolated CD8-positive T cells in a sterile 5-milliliter polystyrene round-bottom tube for 1 hour at 4 degrees Celsius with continual mixing.
At the end of the incubation, wash the nanoparticle cell suspension three times on the magnetic stand as demonstrated, re-suspending the artificial antigen-presenting cells and the enriched CD8-positive T cells in 500 microliters of fresh supplemented medium with 1% T cell growth factor, after the last wash.
For maximum cell recovery, it is critical to leave the particles in cells on the magnetic column for at least 2 minutes, and to aspirate the buffer carefully without disrupting the particles and cells subject to the magnetic field.
After counting, seed 2.5 x 105 enriched CD8-positive T cells and magnetic particle mixture per 160 microliters of supplemented medium plus 1% TCGF in a 96-well U-bottomed plate. On day three, feed the cells with 80 microliters of supplemented medium with 2% T cell growth factor per well, and return the plate to the cell culture incubator for four more days.
On day seven, harvest the stimulated cells into a 5-milliliter round-bottom tube for counting, and collect the cells by centrifugation. Resuspend the pellet in 0.5 milliliters of PBS supplemented with 0.05% sodium azide and 2% fetal bovine serum for counting, and aliquot 5 x 104 to 5 x 105 cells into new 5-milliliter round bottom tubes for antigen-specific staining.
Label the appropriate tubes with biotinylated MHC-Ig and anti-mouse CD8a for 1 hour at 4 degrees Celsius, followed by a centrifuge wash in fresh PBS, to remove any excess biotinylated immunoglobulin. Then, stain the samples with an appropriate streptavidin-conjugated secondary antibody, and an appropriate live/dead fixable dead cell stain for 15 minutes at 4 degrees Celsius, and read the cells on a flow cytometer to determine the specificity and number of the antigen-specific CD8-positive T cells, according to standard protocols.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

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鼠标朴素CD4