Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
Uploaded: 2018-05-31T13:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

下面的协议指的是鼠标 MoDC 的隔离, 但总体原则可能也适用于其他 DC 子集单元。12-16 周大的 C57Bl/6j 小鼠被维持在美国的实验室动物 Care–accredited 动物设施认证协会。所有议定书均经斯坦福大学和特拉维夫大学机构动物护理和使用委员会批准。
1. 肿瘤相关单核细胞源性 DC 的分离
<ol><li>在层流罩中, 从 CO2安乐死小鼠中取出肿瘤, 并将肿瘤放置在没有胎牛血清的 RPMI 培养基中。 注意: 强烈建议在切除肿瘤之前, 用70% 乙醇喷出老鼠, 并将其喷洒, 以尽量减少毛皮的潜在污染物。确定肿瘤不超过 25-40 毫米2 , 因为较大的肿瘤有更少的 DC, 往往是脆弱的, 容易遭受细胞死亡。如果需要更多的 DC, 每只老鼠都可以在多个地点注射肿瘤细胞。</li>
	<li>在无菌层流罩下, 用手术剪刀将肿瘤劈成小块 (大约 1 x 1 毫米2)。<ol>
<li>将所有肿瘤片段从一只老鼠添加到一个30毫升无菌的扁平底管与磁性搅拌棒, 其中包含5毫升的汉克的平衡盐溶液 (HBSS), 2 毫克/毫升胶原酶 IV, 0.01 毫克/毫升 DNase i。 注意: 髓细胞通过糖基化产生能量, 即使在稳定状态下也是如此。因此, 只使用含有葡萄糖的培养基 (如 HBSS、RPMI 和 DMEM), 因为葡萄糖饥饿的量只有 10-15 分钟, 在24小时内会导致细胞死亡和凋亡. 只使用低内毒素胶原酶 IV, 胶原酶是由革兰氏阳性产生的细菌 (histolyticum 梭菌)。</li>
	</ol>
</li>
	<li>在37个oC 孵化器中搅拌约 200-400 rpm, 用磁力搅拌器进行20-30 分钟。</li>
	<li>添加5毫升完整的介质, 并大力重新悬浮。</li>
	<li>过滤细胞通过一个70µm 细胞过滤器。离心机位于 4 oC 400 区域合作框架内, 5-10 分钟颗粒状细胞。 注意: 不要试图在酶消化后直接分离细胞, 因为有些肿瘤是坏死的, 含有相对较多的细胞碎片或细胞外基质。在15% 密度梯度介质上应用细胞, 只获得细胞。</li>
	<li>悬浮9毫升密度梯度介质与1毫升 10x PBS 调整渗透压, 并获得 100% Percoll 的库存解决方案。将1.5 毫升密度梯度介质溶液与8.5 毫升 HBSS 混合, 并与肿瘤衍生细胞颗粒混合。在室温下, 在15% 密度梯度介质的顶部轻轻地层2毫升的 DMEM, 在400个区域合作框架内离心机为20分钟。丢弃上清液, 因为细胞会在管子的底部形成一个小球。<ol>
<li>两次清洗颗粒细胞, 重新悬浮在10毫升 HBSS 含有2% 的血清, 1% 青霉素/链霉素, 10 毫米 EDTA (隔离缓冲), 离心机在4个oC 400 区域合作框架为5-10 分钟颗粒细胞。</li>
		<li>在光镜下用台盼蓝计数 hemocytometer 上的细胞。</li>
	</ol>
</li>
	<li>在 1 mL 隔离缓冲区中重新挂起 1 x 108单元格, 并在4个oC 处孵化它们, 并将 CD11b+共轭磁性珠的 30 ul 作为15分钟。 注意: 避免使用 CD11c-conjugated 珠, 因为这会激活 DC 并诱发细胞死亡。不要试图阻止 FcγRII 和 FcγRIII 与 anti-CD16/32 抗体。阻断抗体结扎 FcγR, 诱导强磷酸化的 MAP 激酶和素数的 DC。<ol>
<li>在 4oC 中, 在400区域合作框架中添加9毫升的隔离缓冲和离心细胞, 以消除多余的未绑定珠子。</li>
	</ol>
</li>
	<li>在1毫升隔离缓冲器中吸入上清并重新悬浮细胞。将单元格应用于水洗磁柱上。用3毫升的隔离缓冲器将柱面清洗两次。<ol>
<li>从磁铁中移除该列。将6毫升的隔离缓冲器放到柱上, 通过推柱塞将磁性标记的细胞冲出无菌的收集管中。在 4 oC 上, 400 个区域合作框架内的离心细胞为 5-10 分钟。</li>
		<li>在 100 ul 每 1 x 107单元格中重新暂停单元格, 并使用以下荧光共轭抗体进行染色: 宗族阴性 (TCRb、Siglec F、B220、CD19、FceRI 和 Ly6G)、MHCII、Ly-6C。</li>
	</ol>
</li>
	<li>通过浇小细胞, 使用侧向散射 (SSC) 和前向散射 (FSC) 和完全培养基中的培养基来对细胞进行排序, 并辅以 5 ng/毫升的 GM-脑脊液。孵育细胞1小时在 3 7 oC 为了允许巨噬细胞黏附板材。然后, 将松散的和非黏附的细胞转移到新的培养皿中。 注意:鼠标 MoDC 被定义为 CD11b+/CD11c+/MHCIIhi/Ly6C/int 7, 26, 27.Ly6C 的表达可能在肿瘤模型之间有显著差异, 在某些模型中 (如 LMP) MoDCs 完全缺乏 Ly6C 表达。总的来说, 一个100毫米3 B16F10 肿瘤通常包含 5 x 104 DC, 导致大约 4-5 x 106总细胞。在这些细胞中, 10-15% 是免疫细胞, 8-10% 是 MoDC。增加 DC 数可以通过在多个地点注射肿瘤小鼠来实现。只对小的 SSC/FCS 细胞进行排序, 因为其他髓细胞可以表达诸如 CD11c 和 Ly6C 等标记。 可选: 将肿瘤浸润单核细胞分类为小 SSC/FSC 单元, 表达 Ly6Chi , 对 MHCII 是阴性的。之后, 培养的单核细胞体外与 50 ng/毫升 GM 脑脊液获得 DC。</li>
</ol>2. 从外周血中分离单核细胞源 DC
注意: 由于成熟的 MoDCs 在小鼠血液中比较少见, 下面的协议是指它们从排列的单核细胞中的体外派生。
<ol><li>为了增加血液中单核细胞的水平, 麻醉小鼠与4% 异氟醚, 并注射他们皮下 (南卡罗来纳州) 与1微克的 GM 脑脊液在 50 ul PBS。</li>
	<li>1-2 小时后, 使用 CO2来牺牲鼠标。 注意: 不要因为宫颈脱位而牺牲老鼠, 因为这会导致内出血。</li>
	<li>euthanization 后立即用70% 乙醇喷雾鼠标, 用手术剪刀将覆盖心脏的皮肤取出, 在无菌层流罩下。用乙醇清洁剪刀, 并切断心脏的右心房。慢慢地, 用20毫米 EDTA HBSS 用10毫升注射器和25克针将心脏通过右心室冲洗。<ol>
<li>用无菌注射器从胸腔内收集血液, 将其注入含有硫酸硫酸和 EDTA 的无菌管。继续冲洗心脏, 直到肝脏和肺部变成粉红色或白色。</li>
	</ol>
</li>
	<li>将血液应用到密度梯度 mediumand 离心机在400个区域合作框架在室温下的15分钟低刹车。 注意: 使用无内毒素密度梯度培养基 (通常小于0.12 的欧盟每毫升)。<ol>
<li>将单个核细胞收集到新的试管中。在10毫升 HBSS 中重新悬浮, 其中含有2% 的血清, 1% 青霉素/链霉素和10毫米 EDTA (隔离缓冲器), 然后离心在 4 oC 400 区域合作框架内以5-10 分钟的粒细胞。 警告: 仅使用含有葡萄糖至少1克/升的介质。</li>
	</ol>
</li>
	<li>对于 CD11b 正选择, 请在1毫升隔离缓冲区中重新挂起 1 x 108单元格, 并在 15 oC 上将其孵化为50分钟, CD11b-conjugated 磁性珠子的 4 ul。<ol>
<li>在 4 oc 中, 通过在400区域合作框架中添加9毫升的隔离缓冲和离心来清洗多余的珠子, 使其吸入上清液并在1毫升隔离缓冲区中重新挂起单元格。根据制造商的说明, 在水洗磁柱上应用单元格。</li>
		<li>将该列从磁铁中取出, 将6毫升的隔离缓冲液移到柱上, 然后通过推柱塞将磁性标记的细胞冲出无菌的收集管中。400个区域合作框架内的离心细胞为5-10 分钟, 4 oc. 用3毫升隔离缓冲器清洗该列两次。</li>
		<li>在 1 x 107 cells/100 ul 隔离缓冲区中重新挂起单元格, 并使用以下荧光共轭抗体进行着色: 0.1 微克 CD115、0.25 微克 MHCII 和0.1 微克 Ly-6C/1 x 106细胞。</li>
	</ol>
</li>
	<li>通过在小的侧向散射 (SSC) 和小正散射 (FSC) 单元格上的浇口对单元格进行排序。 注: 鼠炎症单核细胞一般定义为 CD115+/MHCIIlo/阴性/Ly6Chi, 而巡逻单核细胞则定义为 CD115+/MHCIIlo/阴性/Ly6C阴性 45。如果不需要两个子集之间的分离, 则可以排序总的 SSClo/FCSlo/CD115+/MHCII劳单元格。无论是天真的还是含肿瘤的小鼠, 都含有大约 5-8 x 104 MoDC 每1毫升的血液和多达 4-5 x 105 MoDC 注射了 GM-CSF 后。其中, 大约70% 个是炎症单核细胞, 30% 个在单核细胞内巡逻。</li>
	<li>在完全介质中, 将细胞集中在 1 x 106细胞/ml 上, 辅以 20 ng/毫升 GM-CSF。1天后, 将不粘附的和松散黏附的细胞转移到新的板块和文化中, 再增加4-5 天。 注意: 直流介质不应辅以丙酮酸。</li>
</ol>3. 肿瘤 IgG 免疫复合物的制备
<ol><li>培养肿瘤细胞在 75 cm2培养瓶到70% 汇合在完全 DMEM 媒介。<ol>
<li>添加2毫升0.25% 胰蛋白酶/EDTA 分离细胞从培养瓶和监测细胞形态学下的光显微镜, 以避免过度 trypsinization。</li>
		<li>添加8毫升完整培养基 (每2毫升胰蛋白酶), 以抑制胰蛋白酶消化, 离心机在4个oC, 400 区域合作框架, 以5-10 分钟的颗粒细胞。 注意: 请确保使用商业 PCR 试剂盒检查支原体的肿瘤细胞。使用鲎鲎裂解法 (拉尔) 化验28) 检测革兰阴性菌和真菌内毒素的存在。血清必须经过0.22 微米的过滤和测试9联邦法规病毒代码。此外, 通过在 LB 琼脂或肉汤上滴下 100-200 ul, 并在 37 oC 中培养2天, 测试培养基和血清。</li>
		<li>再次将细胞从胰蛋白酶和血清中重新悬浮在10毫升 PBS 和离心机中, 在400个区域合作框架内, 5-10 分钟. 吸出上清, 再重复洗涤2次。</li>
	</ol>
</li>
	<li>在室温下将1.8% 缓冲多聚甲醛中的细胞固定为10分钟。<ol>
<li>用10毫升 PBS 和离心机在4个oC 400 区域合作框架中重新悬浮以 5-10 分钟来清洗细胞。</li>
		<li>吸上清液和重复洗两次。 可选: 对于肿瘤摄取化验, 细胞可以标记为5分钟, 在 37 oC 在 PBS 含有1微米羧基琥珀酰亚胺酯 (CFSE)。CFSE, 然后用完整的介质淬火10分钟冰。在含有2% 血清的 PBS 中, 应广泛冲洗细胞以去除残留染料。</li>
	</ol>
</li>
	<li>重新悬浮细胞 (PBS) 缓冲 (公共广播辅助与 2% FCS + 5 毫米 EDTA) 和0.5 微克/毫升的 anti-CD16/32, 以阻止潜在的非特异蛋白蛋白相互作用。在 U 形96井/盘中的板材, 浓度为每 100 ul 1 x 105单元格。<ol>
<li>添加不同稀释的肿瘤结合抗体, 范围从5微克-5 ng/1 x 105细胞不等。把盘子在冰上孵化15-20 分钟。 注: IgG 抗体从天真的20-24 周大的雌性小鼠血清中分离出来的蛋白质 A 列, 如24所述。</li>
	</ol>
</li>
	<li>通过添加 150 ul 的 PBS 和离心 4 oC 400 区域合作框架中的板以 5-10 分钟来洗涤细胞。<ol>
<li>扔掉上清液, 重复洗两次。在含有荧光共轭二次抗体的 ul 100。在冰上孵化20分钟。</li>
		<li>通过增加 200 ul 的离心, 在400个区域合作框架中添加 5-10 分钟, 以洗涤细胞. 丢弃上清液并重复清洗。</li>
		<li>用流式细胞仪分析肿瘤的结合度, 确定细胞所需的最小浓度。 注意: 不应在随后的功能检测中使用不增加染色瘤细胞平均荧光强度 (同种) 的抗体。</li>
	</ol>
</li>
</ol>4. 用肿瘤 IgG 芯片活化 MoDC
<ol><li>涂层肿瘤细胞的 IgG 浓度最低, 如3.1 节所述, 通过3.4。3<ol>
<li>在激活 MoDC 与肿瘤 IC 的前一天, 取代 MoDC 培养基, 其中含有 GM-脑脊液。要做到这一点, 轻轻地吸入介质和洗涤细胞一次与预热完整的文化媒体。 注: 分离成熟的肿瘤相关 MoDC 应培养至少 2-3 小时 (甚至一夜之间) 后, 在完全的媒体排序, 没有 GM 脑脊液, 并在激活他们与 IC。</li>
		<li>对于肿瘤摄取分析, 将 CFSE 标记的肿瘤 IC 添加到 MoDC 中, 比例为 1:5 (IC: MoDC), 并在每 1 x 106 DC 的1毫升完整介质中, 隔夜孵化 12-16 小时。</li>
		<li>对于 MoDC 活化实验的血管化分析, 增加肿瘤 IC 在 1:1 (ic: MoDC) 比和孵化过夜 12-16 小时。 注意: 强烈建议包括一个积极的控制好, 其中 MoDC 刺激与1微克/毫升的 LPS 或其他 TLR 激动剂。</li>
		<li>在夜间激活后, 用隔离缓冲剂轻轻地吸入上清液和洗涤细胞三次, 或10毫米 EDTA HBSS。</li>
		<li>分离 DC 从板, 孵化细胞为 2-3 分钟在1毫升 HBSS 包含10毫米 EDTA, 并且分离细胞由剧烈的吹打。</li>
		<li>400个区域合作框架的离心细胞和 1 x 106 DC 在 90 ul PBS 补充了2% 个 FCS, 5 毫米 EDTA (资产管制署缓冲区) 和0.5 微克的阻断抗体。在冰上孵化 5-10 分钟。</li>
		<li>添加 10 ul 的染色抗体混合物的细胞和孵化在冰上15分钟。</li>
		<li>在4个oC 400 区域合作框架中, 向单元格和离心机中添加2毫升的流式缓冲区, 以 5-10 分钟。</li>
	</ol>
</li>
	<li>重新挂起 200 ul 资产管制器缓冲区中的单元格。<ol>
<li>在运行样品之前, 添加 0.5-1 微克/毫升 DAPI 1-2 分钟, 以排除死细胞的分析。不要过度孵化 DAPI, 因为 MoDC 会在 10-15 分钟内把它拿起来。</li>
	</ol>
</li>
</ol>

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所有的作者都宣称他们没有利益冲突, 也没有什么可透露的。

鉴于接种疫苗的小鼠所需的 dc 数量很大 (大约 2-4 x 106 dc 每一只老鼠), 大多数小鼠的疫苗接种策略都是基于从 BM 和脾脏中分离出的 dc 和他们的前体激活。然而, 试图激活肿瘤 DC在体内, 使用相同的条件, 激活脾脏和 BM DC, 往往是不成功的产生有效的免疫力。在随后的两个出版物中, 卡米et al。发现, 血液和肿瘤 MoDC 有明显的差异, 从脾脏和 BM DC, 鉴于他们承担自然高的内在?...

自发现以来, 树突状细胞 (DC) 一直是广泛研究的焦点, 由于其独特的能力, 扭曲 T 细胞分化1。在过去几十年中, 广泛的研究工作试图定义各种 DC 子集及其在肿瘤进展和免疫中的作用2。DCs 由不同的细胞群组成, 它们在模式识别受体、组织分布和迁移和抗原表示能力方面各不相同,3,4,5。与其他 DC 子集相比, 单核细胞衍生 DC (MoDC) 在肿瘤中的丰富程度更大, 可以很容易地从循环或肿瘤浸润的单核细胞6,7中生成。因此, 许多试图利用其相对患病率的临床试验都是基于体内和体内操作自体 MoDC, 以诱导 T 细胞免疫89。
同样, 以 DC 为基础的实验性肿瘤模型接种需要2-3 系列注射, 5-7 天间隔, 1-2 x 106激活 DC 脉冲与肿瘤抗原。因此, 为了实现这一大数目的 DC, 大多数老鼠的研究主要使用 MoDC 培养的骨髓 (BM) 前体在 GM-CSF 7-9 天 (IL-4 是不需要在鼠标设置)10,11。尽管如此, 鉴于 GM-CSF 挖空小鼠有整体正常 DC 隔间12,13, 并考虑到从该区域性获得的混合种群,14这些 DC 的生理相关性已被质疑。
或者, DC 可能经常被隔离从脾脏细胞。然而, DC 只包含大约 0.3-0.8% 的总脾脏细胞 (导致大约 7 x 105 dc 或脾脏) 和这些细胞, 只有 CD103+ DC 和 MoDC 可以迁移回到淋巴器官。由于 MoDCs 包括大约10-15% 的脾脏 DC 人口15,16, 大多数隔离协议产生大约 1 x 105 MoDC 每脾脏。MoDC 的扩张可以通过注射转染 B16 细胞分泌 GM-脑脊液来实现, 导致脾脏 MoDC17增加100倍。然而, 使用 MoDC 开发 DC 疫苗是有限的, 因为这个程序不能在人类和获得的 MoDC 已经高度激活。
除了获得足够数量的 dc, 开发有效的 dc 疫苗抗自体癌细胞的另一个挑战是缺乏足够的危险信号在肿瘤的设置, 以充分激活 dc。诱导的共同刺激信号通常是通过激活模式识别受体 (PRR), 或 c 型凝集素信号通路18,19,20,21。另外一种激活 DC 的方法利用它们通过与表面 Fcγ受体 (FcγR) 的相互作用来处理抗原的能力。事实上, 一些重要的手稿表明, 注射 MoDC 的 BM 前体激活的肿瘤 IgG 芯片可以防止肿瘤生长的预防性设置, 并可导致根除已建立的肿瘤22,23.
在最近的两篇论文中,卡米等发现, 与 BMDC 和脾 DC 相反, MoDC 从血液和肿瘤不能反应的 IgG IC 没有额外的刺激。发现这是由于存在高胞内水平的酪氨酸磷酸调节 FcγR 信号24,25。通过在 dc 中定义一个关键的检查点, 这项工作提供了对成功的基于 dc 的疫苗接种要求的重要洞察力。需要额外的刺激, 以使 FcγR 信号, 并推测信号从其他凝集素受体使用类似的磷酸化级联, 从而强调了避免在其孤立的 DC 启动的必要性。
因此, 本议定书描述了从血液和肿瘤 MoDC 的分离, 这与 BM 和脾脏 DC 明显不同, 并强调了在这一过程中值得考虑的预防措施。

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE-Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>OptiPrep</td><td>StemCell Technologies</td><td>07820</td><td></td></tr><tr><td>CD45 MicroBeads</td><td>Miltenyi</td><td>130-052-301</td><td></td></tr><tr><td>EasySep Monocyte Isolation Kit</td><td>StemCell Technologies</td><td>19861</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C9697-50MG</td><td>Test each lot for endotoxin</td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25-10MG</td><td></td></tr><tr><td>HBSS</td><td>ThermoFisher</td><td>14025092</td><td></td></tr><tr><td>FBS</td><td>ThermoFisher</td><td>16140071</td><td>Test each lot for endotoxin</td></tr><tr><td>PE-CD11c</td><td>Biolegend</td><td>117307</td><td></td></tr><tr><td>APC-CD11b</td><td>Biolegend</td><td>101211</td><td></td></tr><tr><td>Brilliant Violet 650 MHCII</td><td>Biolegend</td><td>107641</td><td></td></tr><tr><td>AF48- CD86</td><td>Biolegend</td><td>105017</td><td></td></tr><tr><td>APC/Cy7-Ly-C6</td><td>Biolegend</td><td>108423</td><td></td></tr><tr><td>PE/Cy7-CD15</td><td>Biolegend</td><td>135523</td><td></td></tr></tbody></table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

树突状细胞 (DC) 是不同的细胞群, 它们的细胞膜标记物, 迁移模式和分布, 并在其抗原表示和 T 细胞活化能力。由于大多数实验性肿瘤模型的接种都需要数以百万计的 DC, 所以它们被广泛地从骨髓或脾脏中分离出来。然而, 这些 dc 明显不同于血液和肿瘤 DC 在他们的反应免疫复合物 (IC), 大概是其他 Syk 耦合凝集素受体。重要的是, 鉴于 dc 对危险相关分子的敏感性, 在隔离步骤之一中交联激活受体的内毒素或抗体的存在可能导致 DC 的启动, 从而影响参数, 或者至少剂量, 需要激活他们。因此, 我们在这里描述一个详细的协议, 以隔离 MoDC 从血液和肿瘤, 同时避免其过早活化。此外, 还提供了一种用于肿瘤 IC 的 MoDC 活化的协议及其后续分析。

我们最初比较了来自天真自体和同种异体小鼠的抗体与肿瘤细胞的结合能力。为此, B16F10 和 LMP 肿瘤细胞系固定在多聚甲醛和广泛洗涤。B16F10 是黑色素瘤细胞系, 最初是从 C57Bl/6 小鼠的肺转移中分离出来的。LMP 是从 KrasG12D/+、LSL-Trp53R172H/+ 和 Pdx-1-Cre 小鼠中分离出来的胰腺肿瘤细胞, 在129F1 小鼠体内生长稳定。为了获得 IC, 肿瘤细胞在冰上孵化20分钟, 每 1 x 105肿瘤细?...

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

单核细胞衍生的 DC (MoDC) 可以感觉到少量的危险相关分子, 因此很容易被引。我们提供了一个详细的协议, 以隔离 MoDC 从血液和肿瘤和他们的活化与免疫复合体, 同时强调应考虑的关键预防措施, 以避免其过早激活。

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

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

isolation-protocol-mouse-monocyte-derived-dendritic-cells-their

Research

JoVE Journal

Cancer Research

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

11.3K 次观看.  Tel-Aviv University. 单核细胞衍生的 DC (MoDC) 可以感觉到少量的危险相关分子, 因此很容易被引。我们提供了一个详细的协议, 以隔离 MoDC 从血液和肿瘤和他们的活化与免疫复合体, 同时强调应考虑的关键预防措施, 以避免其过早激活。

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

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

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 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Isolation and Activation of Murine Lymphocytes

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

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

分离及小鼠淋巴细胞的激活

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

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

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 ...

使用唾液酸酶处理从分离的 PBMC 中去除唾液酸化单核细胞衍生的 DC

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

作者聚焦:使用基于唾液酶的细胞表面酶处理树突状细胞成熟 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

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小鼠肺树突状细胞的分离

小鼠树突状细胞亚群的分离的高效和高产的方法

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

分离及小鼠淋巴细胞的激活

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

从原发肿瘤的免疫细胞的分离

一种新的生理免疫原树突状细胞生成方法

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

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成