我们感谢西蒙博士的英语编辑。这项研究得到了干杯研究所 (ISCIII) 的赠款的支持 (PI14/00526;PI17/01395;CP11/00145;CPII16/00022), 米格尔 Servet 计划和基金会拉蒙 Areces;Fondo Europeo de 收复与发展区域 (菲德) 共同出资。CNIC 得到经济、工业和竞争力部 (经济) 和 CNIC 基金会的支持, 是 Severo 奥乔亚卓越中心 (SEV-2015-0505)。RTF 是由基金会拉蒙 Areces 和 CNIC, VZG 由 ISCIII, BHF 由 Investigación 疗养院医院 12 de Octubre (imas12) 和 JMG G ISCIII 的 Servet 计划和 imas12。

根据西班牙和欧洲准则, 基金会 Investigaciones Cardiovasculares 卡洛斯 III (CNIC) 和 Comunidad 国立马德里批准了实验程序。小鼠在特定病原体游离 (SPF) 条件下繁殖, 并通过二氧化碳 (CO2) 吸入进行安乐死。
1. 从胫骨和股骨中分离小鼠骨髓细胞
注: C57BL/6 同类系小鼠菌株携带的差异白细胞标记Ptprca, 一般认为 CD45.1 或 Ly5.1, 而野生型 C57BL 菌株携带Ptprcb 等位基因, 称为 CD45.2 或 Ly5.2。CD45.1 和 CD45.2 变异可以用抗体的流式细胞术来区分。CD45.1、CD45.2 和 CD45.1/CD45.2 小鼠可作为细胞来源或接受移植, 允许通过流式细胞仪追踪不同细胞群。优先使用年龄和性别匹配的雄性或雌性小鼠低于12周的年龄。
<ol><li>
股骨和胫骨的制备
	<ol>
<li>弄死老鼠使用由机构动物保育委员会批准的议定书。</li>
		<li>用70% 乙醇喷洒动物表面, 对后肢进行消毒。</li>
		<li>使用无菌剪刀, 镊子和手术刀。用手术刀, 做一个切口在皮肤和去除皮肤从老鼠的远端部分包括皮肤覆盖后四肢。剥下小腿肌肉周围的皮肤, 完全去除腿部的皮肤 (图 2A, 2B)。</li>
		<li>用手术刀将股四头肌从股骨中分离出来。Disarticulate 髋关节不折断股骨头。用手术刀从胫骨上取出肌肉 (图 2C, 2D)。将股骨与胫骨分开, 不折断骨末端。</li>
		<li>保持骨骼在一个培养皿中含有10% 胎牛血清 (血清) 和1% 青霉素/链霉素在冰冷的1x 罗斯威尔公园纪念研究所 (RPMI) 1640 中等。</li>
	</ol>
</li>
	<li>
单元格隔离 注: 所有后续步骤必须在一个文化罩和无菌材料, 以避免污染。<ol>
<li>在无菌培养皿中, 用手术刀小心翼翼地切断每根骨头的近端和远端。</li>
		<li>反复冲洗的骨骼与总容积10毫升的温暖完整的 RPMI 培养基 (RPMI + 10% 血清, 2 毫米 EDTA, 1% 青霉素/链霉素, 20 毫米 HEPES, 55 µM 2-巯基乙醇, 1 毫米丙酮酸钠, 2 毫米 l-谷氨酰胺)。用25克针附着在1毫升注射器上, 从两端冲洗骨头。</li>
		<li>将 effluate 转移到装有70µm 尼龙网过滤器的50毫升锥形管上。通过温和搅拌和吹打, 小心地去除碎片和细胞企业集团。</li>
		<li>在室温下, 将细胞悬浮在 250 x g处进行10分钟的离心。</li>
		<li>并用重悬细胞颗粒在1毫升的冷红细胞裂解缓冲 (0.15 米 NH4Cl + 1 毫米 KHCO3 + 0.1 毫米 EDTA, 调整 pH 值7.2 与 1 N HCl, 0.4 µm 无菌过滤, 并储存在4°c)。用人工震动在冰上维持5分钟。</li>
		<li>添加10毫升的冷缓冲 (1x 磷酸缓冲盐水 (PBS) + 2 毫米 EDTA + 2% 牛血清白蛋白), 以灭活和冲洗红细胞裂解缓冲液。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>洗涤细胞与25毫升完全 RPMI 培养基和离心机在 250 x g 5 分钟在4°c。</li>
		<li>并用重悬5毫升完全 RPMI 培养基中的细胞。混合50µL 的细胞悬浮1:1 与台盼蓝。在显微镜下确定计数室中的细胞数。使用公式计算单元格数: 细胞/mL = [(非染色细胞 count/4) x 2 x 1万]12。 注: 此方法产生 40 x 106到 60 x 106骨髓细胞每未感染8到12周老 C57BL/6 老鼠。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
	</ol>
</li>
</ol>2. DC 分化、成熟和抗原加载
<ol><li>
BM 细胞采集
	<ol>
<li>在6井超低附着面板上播种完整的培养基, 每毫升中等 106细胞, 通常在5毫升每井。分化的巨噬细胞将附着在培养板上。12小时后, 转移上清, 其中主要含有单核细胞, 以清洁6井板, 丢弃旧6井板与粘附巨噬细胞。</li>
		<li>为了促进细胞分化, 培养非黏附细胞在 5 x 105细胞每毫升在典型地5毫升每 RPMI 媒介包含 20 ng/毫升商业上获得的重组小鼠 GM 脑脊液在37°c 和5% 二氧化碳 (CO2) 和每日观察。</li>
		<li>每3天, 旋转下来悬浮细胞和收集的黏附细胞与5毫米 EDTA 在 PBS 和旋转下来。结合和并用重悬在 5 x 105细胞/毫升的新鲜培养基含有 20 ng/毫升 rm-脑脊液。</li>
		<li>在8天, 在非组织培养处理的无菌培养皿中, 以 5 x 105细胞/毫升为单位, 在含有 20 ng/毫升的并用重悬和 20 ng/毫升脂多糖 (LPS) 的培养基上采集附着体和分离细胞, 以诱导高 MHC II。, CD80 和 CD86 表达。</li>
		<li>用流式细胞术检查 DC 分化, 如步骤2.2 所示。</li>
	</ol>
</li>
	<li>
流式细胞术的分化和成熟分析。
	<ol>
<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。重复此步骤两次。</li>
		<li>阻断 fc 受体通过孵化悬浮细胞颗粒在小鼠 Fc 受体阻滞剂 (纯化的抗小鼠 CD16/CD32 IgG2b抗体) 15 分钟, 4 摄氏度根据制造商的指示。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>对于每个探针, 并用重悬25万细胞在300µL 流式细胞仪缓冲 (1x PBS + 2 毫米 EDTA + 2% BSA) 在4°c。</li>
		<li>将30µL 的细胞溶液转移到96井板上。</li>
		<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
		<li>在制造商的指示之后, 丢弃上清, 并在每个井中添加30µL 的含抗体缓冲液。用抗体在4摄氏度孵育细胞20分钟。 注: 通常用于 DCs、单核细胞和巨噬细胞的标记为 CD11b、CD64、CD115、CD11c、MHCII、CD80 和 CD86 (图 3)。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬在200µL 每井的冷流式细胞术缓冲器包含标记排除死的细胞 (例如,碘碘化物, 4′-, 6-diamidino-2-苯基吲哚 (DAPI) 或胺反应染料) 在制造商推荐的浓度13。</li>
		<li>通过将样本转移到流式细胞术管并进行流式细胞术分析, 在0、3、6和9天排除死细胞, 从而监测细胞分化。</li>
	</ol>
</li>
	<li>
直流抗原加载。
	<ol>
<li>在8天, 离心机样品在 250 x g为5分钟在4°c 和并用重悬药丸在200µL 媒介 (RPMI + 10% 没有抗生素的血清)。重复此步骤两次。</li>
		<li>用10µg/毫升 OVAp (323-339) 孵化 DCs;ISQAVHAAHAEINEAGR) 每 1 x 107 DCs 在1毫升中等 (RMPI + 1%) 在塑料管为至少30分钟在37°c。使用非 OVAp 加载 DCs 作为负控制条件。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬小球在200µL 完全 RPMI 媒介。重复此步骤两次。</li>
		<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬小球在200µL 媒介 (RPMI + 10%) 在 2 x 106细胞/毫升。</li>
	</ol>
</li>
</ol>3. OTII 小鼠天真 CD4 T 细胞的分离
注: 此方法产生约 100 x 106 spleenocytes 每未感染8至12周老 C57BL/6 老鼠。男性和女性都可以使用。CD4 T 细胞可与脾或淋巴结分离;CD4 T 细胞分别占到这些器官中大约25% 和50% 的细胞。在无菌条件下执行以下步骤。
<ol><li>从 OTII 转基因小鼠中分离腹股沟、腋、臂、颈、肠系膜淋巴结和脾脏 (图 4), 并将其转移到含有10毫升完整 RPMI 培养基的塑料培养皿中。</li>
	<li>用2毫升完整的 RPMI 培养基冲洗细胞过滤器, 从50毫升管中取出。将淋巴结和脾脏转移到70µm 细胞过滤器放置在50毫升管。融汇使用注射器柱塞 (图 5), 并添加中等至15毫升。</li>
	<li>离心样品在 250 x g 5 分钟在4°c 和并用重悬颗粒在15毫升完全 RPMI 媒介。重复此步骤两次。</li>
	<li>对于脾细胞悬浮, 并用重悬细胞颗粒, 并溶解在步骤1.2.5 中所示的红细胞。</li>
	<li>离心机细胞悬浮在 250 x g 5 分钟在4°c。</li>
	<li>用25毫升的完全 RPMI 培养基和离心机在 250 x g处冲洗细胞, 在 RT 上为5分钟。</li>
	<li>并用重悬5毫升培养基中的细胞。混合50µL 的细胞悬浮1:1 与台盼蓝。用显微镜下的计数室确定细胞数。使用公式计算单元格数: 细胞/mL = [(非染色细胞 count/4) x 2 x 1万]12。</li>
	<li>重复步骤2.2.2。</li>
	<li>按照制造商的指示, 孵化细胞与1:800 稀释生物素化抗体 CD8α, IgM, B220, CD19, MHCII (I Ab), CD11b, CD11c, CD44, CD25 和 DX5 30 分钟的冰, 以后来阴性选择 CD4 T 细胞。</li>
	<li>按照制造商的指示, 根据电池的数量和珠子的容量, 计算所需的链亲和素涂层磁性微球的数量和隔离 CD4 T 细胞使用磁性细胞分离器和适当的分离列。</li>
	<li>并用重悬5毫升完整的 RPMI 培养基中的细胞, 并保持它们在冰上, 而计数活细胞如步骤3.7 所述。</li>
	<li>提取 2 x 105细胞, 并检查收集细胞的纯度使用抗 CD4, CD3, B220 和 CD8 荧光抗体在流式细胞仪使用制造商推荐抗体稀释。</li>
	<li>为了染色孤立的天真 CD4/OTII T 细胞, 并用重悬 106细胞500µL 缓冲 (1x PBS + 2 毫米 EDTA + 2% BSA)。准备500µL 缓冲器 (1x PBS + 2 毫米 EDTA + 2% BSA), 其中包含双最终制造商推荐浓度的关键细胞示踪染料。通过缓慢地将细胞示踪液添加到细胞悬浮液中, 混合这两种溶液。在37摄氏度孵化细胞5分钟。</li>
	<li>用5毫升的完全 RPMI 培养基和离心机在 250 x g 处清洗细胞, 在4摄氏度时为5分钟。重复此步骤两次。并用重悬在 1 x 107细胞/毫升的1毫升完全 RPMI 培养基中的细胞。</li>
</ol>4.体内活化、增殖和 Th1 分化试验
注: 生命细胞示踪染料羧基双乙酸琥珀酰亚胺酯 (CFSE) 和其他琥珀酰胺酯基染料, 它们在不同波长激发和发出荧光, 被广泛用于评估流动的淋巴细胞增殖。细胞仪由于其荧光强度高, 寿命长, 变异性低, 毒性低14。
<ol><li>过继转输转移静脉 (静脉注射) 1 x 106活细胞示踪染料染色的天真 CD4/OTII T 细胞100µL 的 PBS 每只老鼠。标记的 T 细胞可以过继转输转移的尾静脉或复古轨道注射15;在这两种情况下, 细胞会回到淋巴器官 (图 6A)。</li>
	<li>一天后, 通过过继转输转移 10万 LPS 成熟的 OVAp 加载 DCs (s.i.) 在脚垫 (图 6B), 对接受者老鼠提出挑战。</li>
	<li>从接受者小鼠那里收获腘淋巴结, 并处理它们, 直到获得单细胞悬浮后, 与先前在3.1 和3.2 中描述的步骤相同。这样做2天后免疫后, 以测量 CD4/OTII T 细胞活化, 并在5天测量增殖加上 Th1 分化。</li>
	<li>为了分析2天的 T 细胞活化, 用荧光抗体对 CD4、CD69 和 CD25 (可选 CD45.1 和 CD45.2) 染色细胞, 并确定 C69 人口中CD25 + CD4+ T 细胞的百分比。用流式细胞仪分析 (图 7)。</li>
	<li>为了检查5天的 T 细胞增殖, 染色细胞使用适当的荧光抗体对 CD4 和可选的 CD45.1 和 CD45.2。用流式细胞仪分析 CD4 种群中生命细胞示踪染料信号的衰减情况 (图 8)。在这些研究中可以确定几个参数, 例如, 细胞分裂的数量由标记与关键细胞示踪染料, 每个荧光峰中分裂细胞的百分比和总细胞的分裂细胞百分比人口。</li>
	<li>为了确定5天的 Th1 分化, 40万个96井板中的200µL 的 RPMI 培养基中有1个µM ionomycin 和 10 ng/毫升佛波12酯13醋酸酯 (PMA), 在孵化箱6摄氏度的37小时。在过去的4小时, 添加3–10µg/毫升 brefeldin A 阻断细胞因子分泌的培养基。</li>
	<li>离心板5分钟在 250 x g在4°c。通过快速反演96井板, 丢弃上清液。</li>
	<li>重复步骤2.2.2。</li>
	<li>在30µL (1x PBS + 2 毫米 EDTA + 2% BSA) 中孵化15分钟, 在4摄氏度处染色细胞膜, 其中含有 CD4 的抗体, 并可选择在制造商推荐的稀释上 CD45.1 和 CD45.2。通过添加150µL 缓冲细胞 (1x PBS + 2 毫米 EDTA + 2% BSA) 和离心板为5分钟在 250 x g在4°c 和放弃上清。</li>
	<li>通过在 RT 中添加100µL 2% paraformaldehyde/1%sucrose 20 分钟来修复细胞. 离心机板材为5分钟在 1000 x g和4°c。放弃上清。</li>
	<li>Permeabilize 细胞增加50µL 胞内通透缓冲 (1x PBS + 0.1% BSA, 0.01 米 HEPES, 0.3% 皂苷) 和孵育30分钟在4摄氏度。</li>
	<li>增加150µL 的 PBS 和离心机5分钟在 1000 x g在 RT. 丢弃上清。</li>
	<li>在缓冲 (PBS + 0.1% 皂苷) 中加入30µL 的荧光共轭抗 IFNγ抗体, 在 RT 中孵化30分钟, 从而染色细胞内细胞因子。</li>
	<li>通过添加150µL 的缓冲 (PBS + 0.1% 皂苷) 来洗涤细胞。将板材离心5分钟, 1000 x g在 RT 上, 然后丢弃上清。重复此步骤两次。</li>
	<li>用流式细胞仪分析细胞数量 (图 8)。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Naive+CD4(+)+T+Cells:+A+Tool+for+Understanding+the+Development+of+Atherosclerosis.">In Vitro Differentiation of Naive CD4(+) T Cells: A Tool for Understanding the Development of Atherosclerosis.</a> Methods in Molecular Biology. 1339, 177-189 (2015).</li><li>Toribio-Fernandez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamin+A/C+augments+Th1+differentiation+and+response+against+vaccinia+virus+and+Leishmania+major.">Lamin A/C augments Th1 differentiation and response against vaccinia virus and Leishmania major.</a> Cell Death &amp; Disease. 9 (1), 9 (2018).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> Journal of Visualized Experiments. (113), (2016).</li><li>Cruz-Adalia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+CD4(+)+T+cells+present+bacterial+antigens+to+induce+cytotoxic+and+memory+CD8(+)+T+cell+responses.">Conventional CD4(+) T cells present bacterial antigens to induce cytotoxic and memory CD8(+) T cell responses.</a> Nature Communications. 8 (1), 1591 (2017).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> Journal of Visualized Experiments. (88), e51627 (2014).</li><li>Cruz-Adalia, A., Ramirez-Santiago, G., Torres-Torresano, M., Garcia-Ferreras, R., Veiga Chacon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cells+Capture+Bacteria+by+Transinfection+from+Dendritic+Cells.">T Cells Capture Bacteria by Transinfection from Dendritic Cells.</a> Journal of Visualized Experiments. (107), e52976 (2016).</li><li>Cossarizza, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+of+flow+cytometry+and+cell+sorting+in+immunological+studies.">Guidelines for the use of flow cytometry and cell sorting in immunological studies.</a> European Journal of Immunology. 47 (10), 1584-1797 (2017).</li><li>Quah, B. J., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+methods+for+measuring+lymphocyte+proliferation+in+vitro+and+in+vivo+using+CFSE-like+fluorescent+dyes.">New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.</a> Journal of Immunological Methods. 379 (1-2), 1-14 (2012).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animals (NY). 40 (5), 155-160 (2011).</li><li>Warth, S. C., Heissmeyer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+transduction+of+naive+CD4+T+cells+to+study+Treg+differentiation.">Adenoviral transduction of naive CD4 T cells to study Treg differentiation.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Liou, H. L., Myers, J. T., Barkauskas, D. S., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+the+mouse+popliteal+lymph+node.">Intravital imaging of the mouse popliteal lymph node.</a> Journal of Visualized Experiments. (60), (2012).</li></ol>

作者没有什么可透露的

该协议允许对 BM 衍生 DCs 的能力进行表征, 以调节天真 CD4 T 细胞的活化、增殖和分化。此外, 它还可以用来评估 CD4 T 细胞的易感性, 由 BM 衍生 DCs。通过该协议, 可以在体内测量这些事件的变化。
根据调查的假设, 可以使用 T 细胞和 DCs 的几种组合。例如, 你可以分析敲或敲出特定基因的后果, 或者 CD4 T 细胞活化、增殖或 Th1 分化的特定刺激的效率。这些过程可以在 DCs 或 ...

CD4 T 细胞和抗原呈现细胞 (apc), 如 DCs 是需要的调解人免疫的微生物病原体1,2。在外周淋巴器官中, CD4 T 细胞在 apc3、4、5所提出的特异抗原识别后激活。活化 CD4 T 细胞增殖和分化为独特的特定效应细胞, 是必要的发展一个正确的适应性免疫应答6,7。控制这些过程是至关重要的, 以产生适当的适应性防御, 杀死病原体而不产生有害的组织损伤8。Th 细胞是根据表面分子、转录因子和效应细胞因子的表达或产生而定义的, 对病原体的反应执行必要和精确的功能1。细胞 Th1 细胞子集表达转录因子 T 打赌和细胞因子干扰素γ (IFNγ) 和参与宿主防御细胞内病原体1。量化 CD4 t 细胞的活化、增殖和 Th1 分化是评估 t 细胞在免疫应答中发挥作用的有用手段。
该协议能够在体内分析体外生成的 BM 衍生 DCs 的能力, 以调节天真 CD4 T 细胞的活化、增殖和 Th1 分化。该议定书还有助于评估幼稚 CD4 T 细胞被激活、诱导增殖和 Th1 分化的能力 (图 1)。这个多才多艺的协议绕过无法具体地操作或选择被研究的细胞人口在纯粹体内协议。利用基因修饰小鼠5的 BM, 或通过治疗或基因操作分离的 bm 细胞9, 可以研究不同分子和处理对 DCs 的影响。同样, t 细胞反应可以探索通过获得 t 细胞为领养转移从不同的来源或在几操作以后3,8,10。
该协议的主要优点有两个方面。用流式细胞仪分析 T 细胞活化、增殖和 Th1 分化;这与体内研究相结合, 从而避免了在体外可能发生的改变, 包括细胞类型和其他仅在完好器官中发现的因素11。
使用重要染料是一种广泛使用的技术来跟踪细胞增殖, 同时避免使用放射性。用这些试剂进行增殖的测量是基于细胞分裂后的染料稀释。此外, 这些染料可以在多波长检测, 并易于分析的流式细胞术与多种荧光抗体或标记。通过流式细胞术分析 T 细胞的活化、增殖和 Th1 分化, 我们强调了该协议的实用性。

<table><tbody><tr><td>Ethanol</td><td>VWR Chemicals</td><td>20,824,365</td><td>5 L</td></tr><tr><td>Scissors</td><td>Fine Science Tools (F.S.T.)</td><td>14001-12</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools (F.S.T.)</td><td>11000-13</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools (F.S.T.)</td><td>11253-20</td><td></td></tr><tr><td>Scalpel</td><td>Fine Science Tools (F.S.T.)</td><td>10020-00</td><td>Box of 100 blades</td></tr><tr><td>Fetal Bovine Serum</td><td>SIGMA</td><td>F7524</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>LONZA</td><td>DE17-602E</td><td></td></tr><tr><td>Roswell Park Memorial Institute medium (RPMI)</td><td>GIBCO</td><td>21875-034</td><td></td></tr><tr><td>Sterile Petri dishes</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>25-gauge needle</td><td>BD Microlance 3</td><td>300600</td><td></td></tr><tr><td>1 ml syringe</td><td>Novico</td><td>N15663</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>70 &amp;mu;m nylon web filter</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>Red blood lysis buffer</td><td>SIGMA</td><td>R7757</td><td>100 Ml</td></tr><tr><td>EDTA</td><td>SIGMA</td><td>ED2SS-250</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>SIGMA</td><td>A7906</td><td>100 g</td></tr><tr><td>Trypan blue</td><td>SIGMA</td><td>302643-25G</td><td></td></tr><tr><td>Culture-plates</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Cambrex</td><td>BE17-737E</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Merck</td><td>8-05740-0250</td><td></td></tr><tr><td>Sodium pyruvate</td><td>LONZA</td><td>BE13-115E</td><td></td></tr><tr><td>L-glutamine</td><td>LONZA</td><td>BE17-605E</td><td></td></tr><tr><td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td><td>Prepotech</td><td>315-03</td><td></td></tr><tr><td>lipopolysaccharide (LPS)</td><td>SIGMA-ALDRICH</td><td>L2018</td><td></td></tr><tr><td>96-well-plate</td><td>Costar</td><td>3799</td><td></td></tr><tr><td>v450 anti-mouse CD11b antibody</td><td>BD Biosciences</td><td>560455</td><td></td></tr><tr><td>PE anti-mouse CD64 antibody</td><td>BioLegend</td><td>139303</td><td></td></tr><tr><td>PE anti-mouse CD115 antibody</td><td>BioLegend</td><td>135505</td><td></td></tr><tr><td>FITC anti-mouse CD11c antibody</td><td>BioLegend</td><td>117305</td><td></td></tr><tr><td>FITC anti-mouse MHCII antibody</td><td>BioLegend</td><td>125507</td><td></td></tr><tr><td>APC anti-mouse CD86 antibody</td><td>BioLegend</td><td>105011</td><td></td></tr><tr><td>APC anti-mouse CD80 antibody</td><td>BioLegend</td><td>104713</td><td></td></tr><tr><td>Flow Cytometry tubes</td><td>Zelian</td><td>300800-1</td><td>PS 12X75 5mL</td></tr><tr><td>OTII Ovoalbumine peptide</td><td>InvivoGen</td><td>323-339</td><td></td></tr><tr><td>anti-mouse biotinylated CD8&amp;alpha; antibody</td><td>Tonbo Biosciences</td><td>30-0081-U500</td><td></td></tr><tr><td>anti-mouse biotinylated IgM antibody</td><td>BioLegend</td><td>406503</td><td></td></tr><tr><td>anti-mouse biotinylated B220 antibody</td><td>Tonbo Biosciences</td><td>30-0452-U500</td><td></td></tr><tr><td>anti-mouse biotinylated CD19 antibody</td><td>Tonbo Biosciences</td><td>30-0193-U500</td><td></td></tr><tr><td>anti-mouse biotinylated MHCII (I-Ab) antibody</td><td>BioLegend</td><td>115302</td><td></td></tr><tr><td>anti-mouse biotinylated CD11b antibody</td><td>Tonbo Biosciences</td><td>30-0112-U500</td><td></td></tr><tr><td>anti-mouse biotinylated CD11c antibody</td><td>BioLegend</td><td>117303</td><td></td></tr><tr><td>anti-mouse biotinylated CD44 antibody</td><td>BioLegend</td><td>103003</td><td></td></tr><tr><td>anti-mouse biotinylated CD25 antibody</td><td>Tonbo Biosciences</td><td>30-0251-U100</td><td></td></tr><tr><td>anti-mouse biotinylated DX5 antibody</td><td>BioLegend</td><td>108903</td><td></td></tr><tr><td>streptavidin-coated magnetic microbeads</td><td>MACS Miltenyi Biotec</td><td>130-048-101</td><td></td></tr><tr><td>Magnetic cell separator</td><td>MACS Miltenyi Biotec</td><td>130-090-976</td><td>QuadroMACS Separator</td></tr><tr><td>Separation columns</td><td>MACS Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>PE anti-mouse CD4 antibody</td><td>BioLegend</td><td>100408</td><td></td></tr><tr><td>APC anti-mouse CD3 antibody</td><td>BioLegend</td><td>100235</td><td></td></tr><tr><td>FITC anti-mouse CD8 antibody</td><td>Tonbo Biosciences</td><td>35-0081-U025</td><td></td></tr><tr><td>Cell Violet Tracer</td><td>Thermofisher</td><td>C34557</td><td></td></tr><tr><td>APC anti-mouse CD25 antibody</td><td>Tonbo Biosciences</td><td>20-0251-U100</td><td></td></tr><tr><td>Alexa647 anti-mouse CD69 antibody</td><td>BioLegend</td><td>104518</td><td></td></tr><tr><td>PerCPCY5.5 anti-mouse CD45.1 antibody</td><td>Tonbo Biosciences</td><td>65-0453</td><td></td></tr><tr><td>APC anti-mouse CD45.1 antibody</td><td>Tonbo Biosciences</td><td>20-0453</td><td></td></tr><tr><td>PECY7 anti-mouse CD45.1 antibody</td><td>Tonbo Biosciences</td><td>60-0453</td><td></td></tr><tr><td>FITC anti-mouse CD45.2 antibody</td><td>Tonbo Biosciences</td><td>35-0454</td><td></td></tr><tr><td>Ionomycin</td><td>SIGMA-ALDRICH</td><td>I0634</td><td></td></tr><tr><td>Phorbol 12 Myristate 13 Acetate (PMA)</td><td>SIGMA</td><td>P8139</td><td></td></tr><tr><td>Brefeldin A (BD GolgiPlug)</td><td>BD</td><td>555029</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Millipore</td><td>8-18715-02100</td><td></td></tr><tr><td>Intracellular permeabilization buffer</td><td>eBioscience</td><td>00-8333</td><td></td></tr><tr><td>APC anti-mouse IFNg antibody</td><td>Tonbo Biosciences</td><td>20-7311-U100</td><td></td></tr><tr><td>Fc-block (anti-mouse CD16/CD32)</td><td>Tonbo Biosciences</td><td>70-0161-U100</td><td></td></tr><tr><td>B6.SJL CD45.1 mice</td><td>The Jackson Laboratory</td><td>002014</td><td></td></tr><tr><td>BD LSRFortessa&amp;trade; Cell Analyzer</td><td>BD Biosciences</td><td>649225</td><td></td></tr><tr><td>DAPI Solution</td><td>Thermofisher</td><td>62248</td><td></td></tr></tbody></table>

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

量化的天真 CD4 t 细胞活化, 增殖, 并分化为 t 帮助 1 (Th1) 细胞是一个有用的方法来评估 t 细胞在免疫应答中发挥的作用。本协议描述骨髓 (BM) 祖细胞的体外分化, 以获得粒细胞巨噬细胞集落刺激因子 (GM-脑脊液) 衍生树突状干细胞 (DCs)。该协议还介绍了蛋白蛋白肽 (OVAp) 负载的 GM-脑脊液衍生 DCs 和天真的 CD4 T 细胞 OTII 转基因小鼠的移植, 以分析体内活化、增殖和 Th1 分化的转移 CD4 T 细胞。这个协议绕过了纯粹的体内方法的限制, 因为无法专门操作或选择所研究的细胞群体。此外, 本议定书允许在体内环境中进行研究, 从而避免对可能发生在体外的功能性因素的改变, 包括细胞类型和其他仅在完整器官中发现的因素的影响。该协议是一个有用的工具, 以产生变化的 DCs 和 T 细胞, 修改适应性免疫反应, 有可能提供重要的结果, 以了解许多免疫相关疾病的起源或发展。

图 1说明了本协议中描述的步骤。图 2展示了小鼠 BM 细胞的分离和培养。在这些培养中加入了 GM-csf 和 LPS, 可使 dcs 的体外生成和成熟.图 3说明了流式细胞术对所获得 dcs 的分化和成熟的分析. OVAp 负载的 GM-脑脊液 BM 基 dcs分离的重要细胞微量染料染色 CD4/OTII T 细胞过继转输转移到小鼠身上。<strong class...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

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

在这里, 我们提出了一种在体内测定由 GM-脑脊液骨髓 (BM) 衍生树突状细胞 (CD4) 诱导的天真的 t 细胞 (t 细胞) 活化、增殖和 Th1 分化的方法。此外, 该协议还描述了 BM 和 t 细胞分离、dc 生成和 dc 和 t 细胞的移植。

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 ...

an-vivo-mouse-model-to-measure-nave-cd4-t-cell-activation

Research

JoVE Journal

Immunology and Infection

19.4K 次观看.  Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). 在这里, 我们提出了一种在体内测定由 GM-脑脊液骨髓 (BM) 衍生树突状细胞 (CD4) 诱导的天真的 t 细胞 (t 细胞) 活化、增殖和 Th1 分化的方法。此外, 该协议还描述了 BM 和 t 细胞分离、dc 生成和 dc 和 t 细胞的移植。

视频: An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

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

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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 ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

医学

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

<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 ...

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

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激活的B细胞CD40的小鼠模型

移植物抗宿主病的诱导和

文化髓系树突状细胞从骨髓前体

代骨髓衍生的双光子成像使用的小鼠树突状细胞

鼠标朴素CD4

活体细胞毒性试验研究肿瘤特异性 CD8⁺ t 细胞反应中的免疫调节

小鼠致耐受性树突状细胞的发育及功能表征

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

小鼠幼稚 CD4⁺ T 细胞体外分化为致病性 Th17 细胞

小鼠幼稚 CD4⁺ T 细胞体外分化为致病性 Th17 细胞