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The Detection of Dendritic Cell Subsets and Maturation in the Tumor Microenvironment by Flow Cytometry   

杨超杨超*金静思金静思*邓刘福邓刘福  (*contributed equally to this work)
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摘要:树突状细胞是一群功能强大的抗原提呈细胞,是联系固有免疫和特异性免疫的中坚力量。cDC表达CD11c和MHCII,可分为cDC1和cDC2两个不同的亚群 (Guilliams等,2014)。cDC1s分化依赖于转录因子BATF3,IRF8和ID2,表达趋化因子受体XCR1;在小鼠肿瘤模型中,一般加用整合素αE分子 (CD103) 来标记 (Salmon等,2016)。而cDC2s的分化依赖于转录因子RELB,IRF4和ZEB2,表面表达CD11b和CD172a分子可使其与cDC1s亚群区分开。cDC1s主要执行交叉提呈肿瘤抗原,激发CD8+T细胞杀伤效应,已被广泛研究证实是肿瘤排异反应及肿瘤免疫治疗的关键亚群 (Bottcher等,2018)。本文阐述如何运用流式技术检测肿瘤微环境内DC亚群及其成熟标志,为相关研究作铺垫。

关键词: 树突状细胞, 肿瘤微环境, 流式技术

材料与试剂

材料:

  1. 眼科剪及眼科镊
  2. 1 ml移液枪及枪头
  3. 6孔细胞培养平底板 (NEST®, catalog number: 032819AA01)
  4. 96孔细胞培养U底板 (NEST®, catalog number: 112518AA03)
  5. 15 ml离心管 (Thermo Fisher Scientific, NuncTM, catalog number: 339650)
  6. 50 ml离心管 (Thermo Fisher Scientific, NuncTM, catalog number: 339652)
  7. 无菌注射器 (上海康德莱企业发展有限公司,KDL®)
  8. 70 μm孔径滤网 (Corning, Falcon®, catalog number: 352350)
  9. 5 ml无菌圆底管 (带滤网) (Corning, Falcon®, catalog number: 352235)

试剂:

  1. 胎牛血清 (Gemini, FoundationTM, catalog number: 900-108)
  2. Anti-FcR (CD16/CD32) 抗体 (BioXCel, catalog number: BE0307)
  3. 胶原蛋白酶I (Worthington Biochemical®, catalog number: LS004186,-20°C储存)
  4. 脱氧核糖核酸酶I,来源于牛胰腺 (Sigma-Aldrich®, catalog number: DN25,-20°C储存) 
  5. DMEM基础高糖培养液 (HycloneTM, catalog number: SH30243.01)
  6. PBS (HycloneTM, catalog number: SH302560.31)
  7. 乙醇
  8. 5% DMEM培养液 (见溶液配方)
  9. FACS buffer (见溶液配方)
  10. 肿瘤组织消化液 (见溶液配方)
抗体:
  1. Anti-FcR (CD16/CD32) (BioXCell, BioXCell, catalog number: BE0307)
  2. Fixable Viability Stain 780 (BD Bioscience, BD, catalog number: 565388)
  3. Anti-mouse-CD45.2-BV785 (Biolegend,  catalog number: 109839)
  4. Anti-mouse-Ly6C-BV605 (Biolegend, catalog number: 128035)
  5. Anti-mouse-Ly6G-BV711 (Biolegend,  catalog number: 127643)
  6. Anti-mouse-CD11b-PE/Cy7 (Biolegend, catalog number: 101216)
  7. Anti-mouse-CD11c-APC (Biolegend,  catalog number: 117310)
  8. Anti-mouse-CD103-APC/Cy7 (Biolegend, catalog number: 121432)
  9. Anti-mouse-F4/80-BV421 (Biolegend, catalog number: 123137)
  10. Anti-mouse-CD86-FITC (Biolegend, catalog number: 105005)
  11. Anti-mouse-MHC class II (I-A/I/E)-PerCP/Cy5.5 (Biolegend,  catalog number: 107625)

仪器设备

  1. 37 °C二氧化碳培养箱 (Thermo Fisher Scientific)
  2. 电热恒温水槽 (上海一恒科学仪器有限公司,model:DK-8AXX)
  3. 低温离心机 (Eppendorf®, model: 5810R)
  4. 微型台式真空泵GL-802B型 (Kylin-bell®, model: GL-802B型)
  5. Flow Cytometer (BD, model: LSRfortessa, or equivalent)
  6. 恒温细胞培养箱
  7. 超净工作台

实验步骤

  1. 前期准备工作
    1.1
    分组标记:根据实验分组需求取若干六孔板,标记荷瘤小鼠信息。六孔板中每孔加入3 ml DMEM高糖培养基,将六孔板置于冰上待用。
    1.2
    配制肿瘤组织消化液:将预先配制的100× 消化液母液 (含100 mg/ml胶原酶I和20 mg/ml DNA酶I) 用含5% FBS的DMEM高糖培养基稀释100倍,即最终工作浓度为1 mg/ml胶原酶I和200 μg/ml DNA酶I。将1× 消化液置于37 °C水浴锅预热待用。
    1.3
    器械准备:根据实验分组需求准备适量的剪刀镊子,用双蒸水洗净后75%乙醇消毒,烘干备用。
  2. 肿瘤组织的分离
  3. 采用颈椎脱臼法将荷瘤小鼠处死,喷洒75%乙醇消毒。左手持弯镊,右手持剪刀在肿瘤右侧1 cm处沿肿瘤边缘剪开一长约2 cm口子。左手捏住剪开处的皮肤向外翻折,可清晰见到肿瘤附着于皮下。用剪刀沿肿瘤边缘轻轻剪开,将肿瘤剥离,注意肿瘤若有溃烂,则避开溃烂之处。将剥离下来的肿瘤组织放入预先准备好的六孔板内。
  4. 肿瘤组织的剪碎
  5. 待全部肿瘤剥离完毕,用移液枪吸出预留DMEM高糖培养基,余大约0.1 ml,以免在剪碎过程中肿瘤组织干燥。手持剪刀小幅度高频率剪碎肿瘤组织,将肿瘤组织剪成泥浆状,肉眼无法看见清晰组织块。注意以上步骤均在冰上操作。
  6. 肿瘤组织的消化
  7. 将肿瘤组织剪成泥浆状后,每孔加入2 ml 1× 消化液,用1 ml枪头将肿瘤组织碎末打散,置于37 °C恒温细胞培养箱消化30 min,每隔10 min将六孔板取出晃动混匀一次。注意严格按照时间消化,以免影响免疫细胞活性。可根据肿瘤大小调整1× 消化液的用量。
  8. 制备肿瘤细胞悬液
  9. 消化完毕后,将六孔板取出放于冰板上,每孔加入4 ml DMEM高糖培养基 (含5% FBS) 终止消化。用巴氏吸管吸取肿瘤组织悬液至70 μm细胞滤网过滤,同时用1 ml注射器后部研磨遗留在滤网上的组织块 (注意研磨时切勿用力,易将脂肪等带入细胞悬液中),用DMEM高糖基础培养基冲洗滤网上的组织块,所得肿瘤悬液4 °C,850 x g,离心5 min。
  10. 染色前封闭
  11. 由于髓系细胞表面表达较多Fc受体,容易对流式抗体有非特异性结合,故采用预先阻断Fc受体封闭。离心后弃上清,根据沉淀多少加入适量FACS缓冲液混匀,取10 μl细胞悬液计数,将悬液细胞密度调至2 × 107个/ml。取100 μl细胞悬液于U底96孔内,按100:3比例加入抗Fc受体抗体 (即每100 μl细胞悬液加入3 μl抗Fc受体抗体),4 °C孵育30 min。
  12. 配制流式染色抗体
  13. 按照表1配制流式抗体混合液 (用FACS缓冲液配制),每孔加入70 μl流式抗体混合液,根据需要计算每个流式抗体所需体积。


注:由于F4/80、MHCII、CD11c、CD86染色均显示漂移,没有明显分群,需要额外增加空白对照 (不加相应的流式抗体) 和荧光扣除对照 (加入与相应流式抗体一样的只带荧光的流式抗体)。以anti-mouse-CD11c-APC为例,空白对照即不加CD11c-APC这支流式抗体,而荧光扣除对照则是添加anti-rat-IgG-APC。

表1. 肿瘤微环境内MDSCs流式染色配色

 注:流式抗体使用配色可自行调整,注意不要使用补偿过大的荧光通道。另外,MC38皮下瘤模型中DC基本为DC2,未见CD103+DC1。

  1. 流式染色
  2. 封闭结束后,4 °C 850 x g离心5 min。弃上清,每孔加入70 μl流式抗体混合液,轻柔吹打混匀,注意不要有气泡。将96孔板置于4 °C避光,染色30 min。
  3. 洗涤、上机
  4. 染色结束后每孔加入200 μl FACS缓冲液,4 °C 850 x g离心5 min。弃上清后再次加入200 μl FACS缓冲液洗涤一次,4 °C 850 x g离心5 min。弃上清,加入300 μl FACS缓冲液重悬细胞,将细胞悬液转移至带滤网的流式管中 (同时过滤细胞悬液)。制备好的流式染色样品用BD Fortessa进行分析,数据处理软件采用FlowJo Version 10.0。


结果与分析

设门方式 (图1):
DC2:Single cell, live, CD45+Ly6C-Ly6G-CD11c+MHCII+F4/80-CD11b+
成熟标志:CD80,CD86

  
图1. 肿瘤内DC的设门方式

溶液配方

注:以下溶液均在超净台内配制。

  1. 5% DMEM培养液
    DMEM基础高糖培养液 + 5%胎牛血清
    4 °C冷藏,1个月内使用
  2. FACS buffer
    PBS + 2%胎牛血清
    4 °C冷藏,1周内使用
  3. 肿瘤组织消化液
    5%DMEM培养液 + 胶原蛋白酶I (工作浓度:1 mg/ml) + 脱氧核糖核酸酶I (工作浓度:200 μg/ml)

参考文献

  1. Bottcher, J. P., Bonavita, E., Chakravarty, P., Blees, H., Cabeza-Cabrerizo, M., Sammicheli, S., Rogers, N. C., Sahai, E., Zelenay, S. and Reis e Sousa, C. (2018). NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control. Cell 172(5): 1022-1037 e1014.
  2. Guilliams, M., Ginhoux, F., Jakubzick, C., Naik, S. H., Onai, N., Schraml, B. U., Segura, E., Tussiwand, R. and Yona, S. (2014). Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol 14(8): 571-578.
  3. Salmon, H., Idoyaga, J., Rahman, A., Leboeuf, M., Remark, R., Jordan, S., Casanova-Acebes, M., Khudoynazarova, M., Agudo, J., Tung, N., Chakarov, S., Rivera, C., Hogstad, B., Bosenberg, M., Hashimoto, D., Gnjatic, S., Bhardwaj, N., Palucka, A. K., Brown, B. D., Brody, J., Ginhoux, F. and Merad, M. (2016). Expansion and activation of CD103+ dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition. Immunity 44(4): 924-938.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:杨超, 金静思, 邓刘福. (2019). 肿瘤微环境内DC细胞亚型和成熟流式检测方法. Bio-101: e1010310. DOI: 10.21769/BioProtoc.1010310.
How to cite: Yang, C., Jin, J. S. and Deng, L. F. (2019). The Detection of Dendritic Cell Subsets and Maturation in the Tumor Microenvironment by Flow Cytometry. Bio-101: e1010310. DOI: 10.21769/BioProtoc.1010310.
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