这项研究得到新加坡国立大学卫生系统 (T1-2014 Oct-12 和 T1-2015 Sep-10) 赠款的支持。

所有在本议定书中使用的老鼠都是在 C57BL/6 的遗传背景下, 并被安置在新加坡国立大学特定的无病原体条件下。所有实验都使用新加坡国立大学机构动物保育和使用委员会批准的协议进行。
1. 试剂和材料制备
<ol><li>制备500毫升磷酸盐缓冲盐水 (PBS), 含2% 胎牛血清 (血清) 和1毫米乙二胺四乙酸 (EDTA)。 注意: 在整个过程中保持缓冲区在冰的最佳效果。</li>
	<li>准备50毫升完整的 RPMI 培养基, 含有 RPMI 1640, 10% 的血清和1% 青霉素-链霉素。使用包含50µM β巯基乙醇的完整 RPMI 培养基进行 THGM 细胞分化。在通风罩中加入β巯基乙醇。</li>
	<li>通过将 anti-CD3e 浓缩的库存稀释到 PBS 中的3微克/毫升, 制备7.5 毫升的 anti-CD3e 抗体混合物。涂层48井板材通过增加 150 ul 抗体混合物对每个井和孵化板材在37°c 至少 1 h, 或者在4°c 隔夜。</li>
	<li>消毒一双剪刀和镊子, 并保持他们在70% 的乙醇夹层解剖。</li>
</ol>2. 小鼠脾的制备
<ol><li>弄死老鼠 (6–8周老) 使用一个制度上批准的 CO2窒息或颈椎脱位。用70% 乙醇喷雾鼠标, 并将其装在其背面的聚苯乙烯块上。将鼠标转移到生物安全柜, 以执行以下步骤。</li>
	<li>用一把钳子握住皮肤, 用一把剪刀将左侧肋骨下面的皮肤切开4厘米。打开腹膜囊露出脾脏, 用无菌钳提取脾脏。</li>
	<li>在50毫升管上放置一个70微米的细胞过滤器, 用2毫升的缓冲器将其预湿。将脾放在细胞过滤器上 (2–3脾一个细胞滤网), 并浸渍使用5毫升注射器柱塞的末端。</li>
	<li>将过滤器和管多次冲洗成细胞隔离缓冲器, 直到所有细胞都冲入管内。丢弃细胞过滤器和离心细胞在 350 x g 5 分钟在4摄氏度。放弃上清。</li>
	<li>并用重悬5毫升冷 (4 °c) 氯化铵-钾 (ACK) 株溶藻缓冲器 (150 毫米 NH4氯) 的细胞颗粒; 10 毫米 KHCO3, 0.1 毫米 Na2EDTA; pH 7.2–7.4), 并将其轻轻地混合为2分钟. 添加10毫升完整的 RPMI 介质以中和应答缓冲, 并立即离心细胞在 350 x g 5 分钟在4摄氏度。离心后要丢弃上清液。</li>
</ol>3. 用磁性 CD4 微珠和细胞分离柱分离 CD4+ T 细胞
<ol><li>并用重悬细胞颗粒在5毫升细胞隔离缓冲。用预分离过滤器 (30 微米) 将细胞悬浮物过滤成新的15毫升管以去除任何碎片。</li>
	<li>采取 10 ul 的细胞悬浮, 并作出10x 稀释通过增加 90 ul 的缓冲。取 10 ul 的稀释细胞悬浮, 并混合它与 10 ul 的台盼蓝的细胞计数。计数 hemocytometer 中的细胞以确定存活细胞的产量。</li>
	<li>离心细胞在 350 x g 5 分钟在4°c 和放弃上清。</li>
	<li>并用重悬细胞在 90 ul 的缓冲每 107细胞。添加 10 ul 磁性 CD4 微珠每 107细胞。在4摄氏度孵化细胞15分钟。为获得最佳效果, 在孵化过程中, 每5分钟轻轻混合一次细胞悬浮。</li>
	<li>当细胞孵化时, 在磁性支架上放置一个分离柱。用2毫升的缓冲器预湿柱。</li>
	<li>在细胞/珠孵化结束时, 用5毫升的缓冲液冲洗细胞, 在4摄氏度时将其离心在 350 x g处5分钟。放弃上清。</li>
	<li>并用重悬1毫升的缓冲细胞, 将样品装入细胞分离柱中, 让它流过。使用15毫升离心管收集 CD4 细胞分数。添加1毫升的缓冲液, 在干燥之前将其冲洗干净。重复洗涤步骤2x。</li>
	<li>从磁支架中取出单元分离柱, 将其放在新的15毫升离心管上。将2毫升的细胞隔离缓冲器添加到细胞分离柱上, 并将柱塞牢固地应用于将细胞从柱上强行取出。</li>
	<li>离心率为 350 x克5 分钟, 在4摄氏度, 以获得 CD4+ 细胞。放弃上清。</li>
</ol>4. 荧光活化细胞分选和 THCD4+ 纯化天真的 CD4+CD25-CD44螺CD62Lhi)
<ol><li>并用重悬在500µL 缓冲液中获得 CD4+ 细胞颗粒。将细胞与含有 CD4-PerCp (2.8 µg/毫升)、CD44-APC (2.4 µg/毫升)、CD25-PE (2 µg/毫升) 和 CD62L-FITC (10 µg/毫升) 的荧光共轭抗体混合物 (表 1) 混合。在冰上孵化细胞以 20–30 min, 同时保护它们不受光照。 注意: 抗体的浓度在实验室中进行了优化。所列出的抗体的数量是来自于小鼠的细胞。</li>
	<li>孵化后, 用5毫升的缓冲液冲洗细胞, 将细胞在 350 x g处进行离心处理, 在4摄氏度时进行5分钟。</li>
	<li>丢弃上清液, 并用重悬500µL 的细胞。使用尼龙网格再次过滤单元格。</li>
	<li>将细胞悬浮液转移到用于细胞分类的转流管。涂型了500µL 缓冲器的采集器管。</li>
	<li>通过 CD4 的分类,获得 CD25CD44 CD62Lhi种群 (天真 CD4+ T 细胞)。门在 CD4+CD25-首先, 然后选择 CD44 CD62Lhi细胞从这个人群。</li>
	<li>将天真的 CD4和 T 细胞分数收集成一个新的15毫升离心管。离心细胞在 350 x g 5 分钟在4°c 和放弃上清。</li>
	<li>并用重悬在含有50µM β巯基乙醇的完整 RPMI 培养基中, CD4+ 的浓度为 106细胞/毫升的天真的 T 细胞。</li>
	<li>在48井板中吸入用于预涂的 anti-CD3e 抗体。种子 25万 (250 µL) 细胞在每个井与 IL-7 (2 ng/毫升), anti-CD28 (1 µg/毫升), 和反 IFNγ (10 µg/毫升) (表 1)。 注意: 细胞因子和抗体的浓度在实验室中被优化为 THGM 细胞分化。</li>
	<li>孵化细胞在37°c 与 5% CO2 3 天。在孵化过程中不要改变培养基。</li>
</ol>5. 小鼠 T 型转基因细胞体外生成的分析
<ol><li>用显微镜检查细胞分化后 3 d 的分化。收获细胞和离心他们在 350 x g 5 分钟在4摄氏度。用完整的 RPMI 介质冲洗细胞2x。 注意: 分化细胞的大小比天真的 CD4+ T 细胞大。</li>
	<li>并用重悬1毫升完整的 RPMI 培养基中的细胞。采取 10 ul 的细胞悬浮和混合它与 10 ul 的台盼蓝, 以确定的细胞数与 hemocytometer。将分化的细胞分成三药剂进行 restimulation 和分析。 注: 细胞内细胞因子染色和 ELISA 检测需要≥0.5 百万细胞, qPCR 分析需要≥100万细胞。</li>
	<li>
为细胞内细胞因子染色, restimulate 细胞与佛波 12-酯 13-醋酸盐 (100 ng/毫升) 和 ionomycin (1 微克/毫升) 存在蛋白质转运抑制剂 (1 µL/毫升) 为第4-6 h。
	<ol>
<li>用 anti-CD4 抗体 (PerCP 共轭, 0.2 毫克/毫升, 1:100 稀释; 或 FITC 共轭, 0.5 毫克/毫升, 1:100 稀释), 以30分钟的方法收割细胞并染色。</li>
		<li>用 200 ul 固定缓冲细胞固定20–60分钟。固定后, 用1毫升的通透缓冲液冲洗细胞2x。</li>
		<li>对 GM 脑脊液 (PE 共轭, 0.2 毫克/毫升, 1:100 稀释), IL-17A (FITC 共轭, 0.5 毫克/毫升, 1:100 稀释) 抗体进行细胞内染色;apc 共轭, 0.2 毫克/毫升, 1:100 稀释), IL-4 (apc 共轭, 0.2 毫克/毫升, 1:100 稀释), 和 IFNγ (apc 共轭, 0.2 毫克/毫升, 1:100 稀释;PE 共轭, 0.2 毫克/毫升, 1:100 稀释) 在黑暗中30分钟。</li>
	</ol>
</li>
	<li>qPCR 分析分化细胞的细胞因子基因表达, 激活细胞与板块绑定 anti-CD3e (3 微克/毫升) (预涂的板块, 如步骤1.3 所述)。在刺激后3小时, 利用苯酚 RNA 萃取试剂, 采集细胞以分离总 RNA, 为 qPCR 制备 cDNA。用于 qPCR 的引物和程序分别列在表 2和3中。</li>
	<li>为分析分化细胞的细胞因子蛋白分泌, restimulate 细胞与板块约束 anti-CD3e (3 微克/毫升) 为 24 h (预涂的板块, 如步骤1.3 所述)。收获细胞培养上清 IL-17 和 GM-脑脊液 ELISA, 以确定其浓度。</li>
</ol>

<ol><li>Zhu, J., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4+T+cells%3A+fates%2C+functions%2C+and+faults%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">CD4 T cells: fates, functions, and faults.</a> Blood. 112, 1557-1569 (2008).</li>
<li>Kara, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailored+immune+responses%3A+novel+effector+helper+T+cell+subsets+in+protective+immunity%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">Tailored immune responses: novel effector helper T cell subsets in protective immunity.</a> PLoS Pathogens. 10, e1003905(2014).</li>
<li>Bou Nasser Eddine, F., Ramia, E., Tosi, G., Forlani, G., Accolla, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+Immunology+meets...Immunology%3A+Modified+cancer+cells+as+professional+APC+for+priming+naive+tumor-specific+CD4%2B+T+cells%5BTitle%5D)+AND+%22Oncoimmunology%22%5BJournal%5D)">Tumor Immunology meets...Immunology: Modified cancer cells as professional APC for priming naive tumor-specific CD4+ T cells.</a> Oncoimmunology. 6, e1356149(2017).</li>
<li>Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TH17+cells+in+development%3A+an+updated+view+of+their+molecular+identity+and+genetic+programming%5BTitle%5D)+AND+%22Nature+Reviews.+Immunology%22%5BJournal%5D)">TH17 cells in development: an updated view of their molecular identity and genetic programming.</a> Nature Reviews. Immunology. 8, 337-348 (2008).</li>
<li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IL-17+and+Th17+Cells%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">IL-17 and Th17 Cells.</a> Annual Review of Immunology. 27, 485-517 (2009).</li>
<li>Sheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((STAT5+programs+a+distinct+subset+of+GM-CSF-producing+T+helper+cells+that+is+essential+for+autoimmune+neuroinflammation%5BTitle%5D)+AND+%22Cell+Research%22%5BJournal%5D)">STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation.</a> Cell Research. 24, 1387-1402 (2014).</li>
<li>Codarri, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RORgammat+drives+production+of+the+cytokine+GM-CSF+in+helper+T+cells%2C+which+is+essential+for+the+effector+phase+of+autoimmune+neuroinflammation%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.</a> Nature Immunology. 12, 560-567 (2011).</li>
<li>El-Behi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+encephalitogenicity+of+T%28H%2917+cells+is+dependent+on+IL-1-+and+IL-23-induced+production+of+the+cytokine+GM-CSF%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.</a> Nature Immunology. 12, 568-575 (2011).</li>
<li>Ivanov, I. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+orphan+nuclear+receptor+RORgammat+directs+the+differentiation+program+of+proinflammatory+IL-17%2B+T+helper+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126, 1121-1133 (2006).</li>
<li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+subset+of+helper+T+cells+promotes+immune+responses+by+secreting+GM-CSF%5BTitle%5D)+AND+%22Cell+Death+and+Differentiation%22%5BJournal%5D)">A novel subset of helper T cells promotes immune responses by secreting GM-CSF.</a> Cell Death and Differentiation. 20, 1731-1741 (2013).</li>
<li>Croxford, A. L., Spath, S., Becher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GM-CSF+in+Neuroinflammation%3A+Licensing+Myeloid+Cells+for+Tissue+Damage%5BTitle%5D)+AND+%22Trends+in+Immunology%22%5BJournal%5D)">GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage.</a> Trends in Immunology. 36, 651-662 (2015).</li>
</ol>

作者没有什么可透露的。

在这里我们描述了一个从小鼠天真的 CD4+ 细胞的体外TH基因分化的协议, 其次是对分化细胞的分析, 以验证该方法。注意, 脾脏和淋巴结均可用于幼稚的 CD4+ t 细胞纯化和 tH基因的分化。细胞内细胞因子染色与流式细胞术相结合的细胞因子表达表明, 在 gm (图 1A) 下, 约有55% 的神经细胞被诱导成为 gm-脑脊液表达细胞。与 th17 分化条件下的细...

CD4+ (tH) 细胞是免疫系统的重要组成部分, 在宿主防御微生物病原体、癌症监测和自身免疫性1、2、3中具有重要作用。在 t 细胞受体 (TCR) 激活, 天真的 CD4+ t 细胞可以被区分为t h1,t h2, th 17, 或调节 t (Treg) 细胞在不同的细胞因子环境传道2,4,5. 最近, 发现了一种主要生产 gm-CSF 的 th细胞的新子集, 并命名为 gm6。IL-7 通过激活信号传感器和转录激活剂 5 (STAT5) 来驱动 THGM 细胞的分化。这些细胞表达了大量的 GM-脑脊液, 同时低表达其他 TH细胞特征因子, 如 IFNγ和 IL-176。在 CD4+ T 细胞介导的 neuroinflammation7、8的发展中, 基因型脑脊液被发现发挥了关键作用。与 IFNγ或 IL-17-expressing 自身反应性 t 细胞相比, 转基因脑脊液表达 t 细胞转移到野生型 (小鼠) 导致早期疾病发作和更高的疾病严重性。此外, Csf2细胞没有诱导实验性自身免疫脑脊髓炎 (EAE) 后被过继转输转移到脑细胞受体, 而缺乏 IFNγ或 IL-17A 的 T 淋巴细胞保留了调解 EAE7的能力。此外, 使用中和抗体对 GM-CSF 的封锁改善了 EAE 疾病严重程度8。此外, 小鼠 t 细胞中 STAT5 的缺失导致了转基因一代的减少, 因此, 在小鼠对 EAE 发展6的抵抗力。这些发现强调了 GM-脑脊液表达 TH细胞在自身免疫性 neuroinflammatory 疾病中的重要性。因此, 建立一种从天真的 CD4+ t 细胞中鉴别 GM-脑脊液表达 tH细胞的方法, 对于研究自身免疫 neuroinflammation 和 T 细胞介导的免疫应答的发病机制具有重要意义。然而, 从小鼠天真的 CD4+ 有效地产生 TH基因电池的协议尚未建立。
在这里, 我们提出了一种方法, 区分小鼠 tHGM 细胞从天真的 CD4+ t 细胞。该协议描述了整个过程, 包括从小鼠中提取脾脏、单细胞悬浮液的制备、CD4 阳性选择、荧光活化细胞分选和 TH细胞。分化和分析。用细胞内细胞因子染色结合流式细胞仪对分化的 T 辅助细胞进行分析, 以确定单细胞水平的细胞因子表达, 采用定量实时 PCR 方法测定 mRNA 水平的细胞因子表达, 并通过 ELISA 对蛋白质水平的细胞因子表达进行评估。该方法可应用于 EAE 等不同条件下的转基因细胞生物学研究中, 其在发病机制中起着重要作用。

<table><tbody><tr><td>RPMI 1640</td><td>Biowest</td><td>L0500-500</td><td></td></tr><tr><td>FBS Heat Inactivated</td><td>Capricorn</td><td>FBS-HI-12A</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>10x PBS</td><td>1st Base</td><td>BUF-2040-10</td><td>Diluted to 1x PBS in sterile water</td></tr><tr><td>EDTA</td><td>1st Base</td><td>BUF-1053-100ml-pH8.0</td><td></td></tr><tr><td>10x ACK buffer</td><td>Biolegend</td><td>420301</td><td>diluted in sterile water</td></tr><tr><td>Cell strainer</td><td>SPL Life Sciences</td><td>93070</td><td></td></tr><tr><td>CD4 microbeads</td><td>Miltenyi Biotec</td><td>130-049-201</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>LS column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>magnetic stand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>1 mL syringe</td><td>Terumo</td><td>SS+01T</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Eppendorf 5810R</td><td></td></tr><tr><td>Sony SY3200 cell sorter</td><td>Sony</td><td>Sony SY3200 cell sorter</td><td></td></tr><tr><td>50 mL conical centrifuge tube</td><td>Greiner Bio-One</td><td>210261</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>FACS tube</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>24 well cell culture plate</td><td>Greiner Bio-One</td><td>662160</td><td></td></tr><tr><td>anti-mouse CD4 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0041-82</td><td></td></tr><tr><td>PE conjugated anti-mouse CD25 (IL-2Ra, p55)</td><td>eBioscience</td><td>12-0251-82</td><td></td></tr><tr><td>anti-human/mouse CD44 APC</td><td>eBioscience</td><td>17-0441-82</td><td></td></tr><tr><td>anti-mouse CD62L FITC</td><td>eBioscience</td><td>11-0621-85</td><td></td></tr><tr><td>Neubauer-improved counting chamber</td><td>Marienfeld</td><td>640010</td><td></td></tr><tr><td>Hyclone Trypan Blue Solution</td><td>GE healthcare Life Sciences</td><td>SV30084.01</td><td></td></tr><tr><td>microscope</td><td>Nikon</td><td>Nikon Elipse TS100</td><td></td></tr><tr><td>Purified&amp;nbsp;anti-mouse CD3e&amp;nbsp;antibody</td><td>Biolegend</td><td>100314</td><td></td></tr><tr><td>Purified hamster anti-mouse CD28</td><td>BD Biosciences</td><td>553295</td><td></td></tr><tr><td>Purified Rat&amp;nbsp;anti-mouse IFN&amp;gamma; &amp;nbsp;</td><td>eBioscience</td><td>16-7312-85</td><td></td></tr><tr><td>Purified anti-mouse IL-4 Antibody</td><td>Biolegend</td><td>504102</td><td></td></tr><tr><td>Recombinant Mouse IL-7 Protein</td><td>R&amp;amp;D system</td><td>407-ML</td><td></td></tr><tr><td>recombinant human TGF-beta</td><td>R&amp;amp;D system</td><td>240-B-010</td><td></td></tr><tr><td>PMA</td><td>Merck Millipore</td><td>19-144</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I0634</td><td></td></tr><tr><td>GolgiPlug protein transport inhibitor</td><td>BD Biosciences</td><td>51-2301KZ</td><td></td></tr><tr><td>Intracellular Fixation&amp;amp;Permeabilization buffer set</td><td>eBioscience</td><td>88-8824-00</td><td></td></tr><tr><td>anti-mouse GM-CSF PE</td><td>eBioscience</td><td>12-7331-82</td><td></td></tr><tr><td>FITC anti-mouse IL-17A</td><td>Biolegend</td><td>506908</td><td></td></tr><tr><td>APC anti-mouse IFN-gamma</td><td>Biolegend</td><td>505810</td><td></td></tr><tr><td>GO Taq qPCR master mix</td><td>Promega</td><td>A6002</td><td></td></tr><tr><td>mouse GM-CSF ELISA Ready-SET-Go!</td><td>invitrogen</td><td>88-7334-88</td><td></td></tr><tr><td>Mouse IL-17A Uncouted ELISA</td><td>invitrogen</td><td>88-7371-22</td><td></td></tr></tbody></table>

in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells

粒-巨噬细胞集落刺激因子 (gm-csf) 生产的 t 帮助器 (tHGM) 细胞是一个新发现的 t 辅助细胞子集, 主要分泌的 GM-脑脊液不产生干扰素 (干扰素) γ或白细胞介素 (IL)-17, 并发现在自身免疫性 neuroinflammation 中起重要作用。从单纯的 CD4+ t 细胞中分离出单纯的脾和 tH基因 CD4+ t 细胞, 是研究 t 细胞介导免疫和自身免疫性疾病的有效方法。这里我们描述了一种方法, 将小鼠天真 CD4+ t 细胞与 IL-7 促进的 tH基因细胞区分开来。用不同的方法分析细胞因子的表达, 包括细胞内细胞因子染色结合流式细胞术、定量实时聚合酶链反应 (PCR), 对分化结果进行评估, 并酶联免疫吸附测定法 (ELISA)。根据这里所描述的 THgm 分化协议, 大约55% 的细胞表达了 IFNα或 IL-17 的最小表达。通过对基因型脑脊液、IFNα和 IL-17在 mRNA 和蛋白质水平上表达的分析, 进一步证实了转基因脑脊液的主要表达。因此, 该方法可用于体外诱导单纯 CD4+ t 细胞与 th基因细胞的分离, 对转基因细胞生物学研究有一定的参考价值。

从两个8周大的雄性 C57BL/6 小鼠中分离出的朴素 CD4+ T 细胞分为三部分。在描述的协议之后, 细胞的一部分被区分成 THGM 细胞。另一部分是在 thgm 条件下培养的 anti-IL-4 抗体 (10 µg/毫升), 以测试 IL-4 封锁的影响, 在分化的 thgm。最后一部分是培养在 TH17 分化条件 (3 µg/毫升 anti-CD3e, 1 µg/毫升 anti-CD28, 10 ng/毫升 TGFβ, 30 ng/毫升 IL-6, 10 µg/毫升反 IFNγ...

Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

在这里, 我们提出了一种区分小鼠粒细胞-巨噬细胞集落刺激因子产生的 t 型CD4+ t 细胞, 包括天真的 CD4+ t 细胞的分离, th基因的分化和差异性 TH基因细胞的分析。该方法可用于转基因细胞的调节和功能研究.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

in-vitro-differentiation-mouse-granulocyte-macrophage-colony

Research

JoVE Journal

Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

7.2K 次观看.  National University of Singapore. 在这里, 我们提出了一种区分小鼠粒细胞-巨噬细胞集落刺激因子产生的 t 型CD4+ t 细胞, 包括天真的 CD4+ t 细胞的分离, th基因的分化和差异性 TH基因细胞的分析。该方法可用于转基因细胞的调节和功能研究.

视频: In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

癌症研究

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

免疫与感染

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

神经科学

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

<meta charset="utf8"></head><body>接触超敏反应小鼠模型中粒细胞-巨噬细胞集落刺激因子-产生 T 辅助细胞的分析

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

接触超敏反应小鼠模型中粒细胞-巨噬细胞集落刺激因子-产生 T 辅助细胞的分析

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

<meta charset="utf8"></head><body>使用流式细胞术从初始 CD4+ T 细胞中分化 Th17 细胞

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>作者聚焦：使用细胞因子浓度调节实现 Th17 细胞的高纯度体外分化

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

小鼠幼稚 CD4+ T 细胞体外分化为致病性 Th17 细胞

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (t_Hgm) 细胞

摘要

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小鼠幼稚 CD4⁺ T 细胞体外分化为致病性 Th17 细胞