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Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs
吞噬实验检测骨髓来源树突状细胞对坏死肿瘤细胞的摄取   

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Abstract

This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.

Background

Studying the phagocytic uptake of necroptotic cells by BMDCs in vitro, is a preliminary step in the examination of immunogenic cell death models (Obeid et al., 2007). Efficient uptake will allow the phagocyte to cross-present antigens to leukocytes and thereby create an immune reaction towards the dead target cells. In this protocol we make use of a CellTrackerTM dye. This type of dye may be toxic in certain concentrations, which may vary depending on which cell type is used. Thus, we recommend users to first find the optimal concentration of the dye for the target cell in use. Optimally, the CellTrackerTM dye itself should not induce any cell death, but should label the target cells so that they become easily separable from the CD11c-positive BMDCs.

Materials and Reagents

  1. Delta treated 10 cm Petri dishes (Thermo Fisher Scientific, NuncTM, catalog number: 153066 )
  2. 15 ml polystyrene centrifuge tubes (Corning, Falcon®, catalog number: 352095 )
  3. 6-well suspension plates (SARSTEDT, catalog number: 83.3920.500 )
  4. 96 V well, 2.0 ml polypropylene plate (Greiner Bio One, MASTERBLOCK®, catalog number: 780285 )
  5. 5 ml round-bottom polystyrene tubes (Corning, Falcon®, catalog number: 352054 )
  6. Bone marrow-derived dendritic cells (BMDCs) isolated from BALB/c WT mice (see Procedure)
  7. CT26 Necroptosis-inducible cells (DD_RIPK3) (Aaes et al., 2016)
  8. Dulbecco’s modified Eagle medium (DMEM), high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 41965062 )
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  10. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
  11. L-glutamine (Lonza, catalog number: BE17-605F )
  12. Roswell park memorial institute (RPMI) 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 52400025 )
  13. 2-mercaptoethanol (50 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  14. Murine GM-CSF (VIB Protein Service Facility, UGent-VIB Inflammation Research Center)
  15. ACK lysing buffer (Lonza, catalog number: 10-548E )
  16. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )
  17. CellTrackerTM green CMFDA dye (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C7025 )
  18. Doxycycline (Sigma-Aldrich, catalog number: D9891 )
  19. B/B homodimerizer (Takara Bio, catalog number: 635059 )
  20. Purified rat anti-mouse CD16/CD32 (Mouse BD Fc BlockTM) (BD, PharmingenTM, catalog number: 553142 )
  21. APC hamster anti-mouse CD11c (BD, PharmingenTM, catalog number: 550261 )
  22. SYTOX® blue nucleic acid stain (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S11348 )
  23. BD FACSuite CS&T Research Beads Kit (BD, FACSuiteTM, catalog number: 650621 )
  24. DMEM CT26 culture medium (see Recipes)
  25. RPMI BMDC/Co-culture medium (see Recipes)
  26. FACS buffer (see Recipes)
  27. Staining solution (see Recipes)

Equipment

  1. Table top centrifuge
  2. BD FACSVerseTM flow cytometer (BD, model: BD FACSVERSE )

Software

  1. BD FACSuiteTM software (BD)
  2. FlowJo software version 10.0.8 or newer (FlowJo)

Procedure

Bone marrow cells were isolated by crushing the femurs and tibias of 7-week-old BALB/c WT mice. Red blood cells were lysed with ACK lysing buffer, and the cells were differentiated into dendritic cells for eight days using RPMI culture medium – 400,000 cells per 2 ml per 6-well. On day three, 2 ml of fresh culture medium was added, and on day six the medium was replaced with fresh culture medium.

  1. Cell death induction in target, CT26 DD_RIPK3, cells
    1. Seed 1 x 106 CT26 DD_RIPK3 cells in 10 ml DMEM culture medium per 10 cm Petri dish.
    2. Wait 5 h for the cells to attach.
    3. Wash the Petri dishes with serum-free DMEM.
    4. Add 10 ml of serum-free DMEM + 1 µM CellTrackerTM green CMFDA to each plate.
    5. Incubate for 30 min at 37 °C.
    6. Remove the medium, and wash the plate with RPMI culture medium.
    7. Add 10 ml RPMI culture medium to each Petri dish, incubate for 2 h at 37 °C.
    8. Induce cell death by adding 1 µg/ml doxycycline + 10 nM B/B homodimerizer.
    9. Incubate for another 18 h at 37 °C.

  2. Co-culture with BMDCs
    1. Collect the BMDCs and CT26 target cells into separate 15 ml Falcon® tubes
      1. Live CT26 cells are collected by adding EDTA (2 ml per 75 cm2), incubating at 37 °C for 2 min, then collecting in RPMI culture medium (8 ml per 75 cm2). Centrifuge at 300 x g for 5 min.
      2. Dead CT26 cells are collected by pipetting only the cells in solution. Centrifuge at 400 x g for 5 min.
    2. Count the cells and resuspend them in RPMI culture medium.
    3. Seed the cells in 6-well suspension plates.
    4. Seed 400,000 BMDCs + 400,000 CT26 cells (ratio 1:1) in a total volume of 4 ml per well.
    5. Incubate for 2 h at 37 °C.
    6. Include wells with cells for single stain and compensation controls:
      1. CT26 cells                                           Non-stained target cell control
      2. CT26 cells – cell death-induced        SYTOX® blue single stain control
      3. CT26 cells                                          CellTrackerTM green single stain control
      4. BMDCs                                               Non-stained BMDC control
      5. BMDCs                                               CD11c-APC single stain control

  3. Immunostaining
    1. Transfer the cells to a deep 96-well plate.
    2. Spin down at 400 x g for 5 min at 4 °C.
    3. Discard the supernatant by flicking the plate, and resuspend the pellet in 200 µl FACS buffer.
    4. Spin down at 400 x g for 5 min at 4 °C.
    5. Resuspend the samples in 200 µl of staining solution (or in single stain solution) with a final antibody concentration (APC-CD11c) of 0.1 µg per 8 x 105 cells.
    6. Incubate for 30 min at 4 °C covered from light.
    7. Wash the samples and spin down at 400 x g for 5 min at 4 °C.
    8. Resuspend in FACS buffer + 1.25 µM SYTOX® blue (or in FACS buffer only for the single stained samples).
    9. Transfer to 5 ml Falcon® round-bottom tubes.
    10. Keep the samples on ice (for maximum 1 h) until acquisition on the flow cytometer.
    11. On the flow cytometer use FACSuiteTM to acquire minimum 50-100,000 events per sample.

  4. Gating strategy in FlowJo
    True uptake of CMFDA-labeled dead cell material by BMDCs is determined using a gating strategy that allows analysis of only single cells and is determined as CD11c+ CMFDA+ double-positive cells expressed as a percentage of all CD11c+ cells. See Figure 1 and Table 1 for the gating strategy and compensation matrix used in FlowJo.


    Figure 1. Gating strategy used for the analysis in FlowJo. Figure 1 shows the gating strategy used, when analyzing the results in FlowJo. Using the forward and side scatter area (FSC-A vs. SSC-A), the debris is excluded from the analysis. In the following two steps, doublet cells are removed, by drawing a gate around the single cells, which roughly gather along the diagonal of FSC-A vs. FSC-H, followed by SSC-A vs. SSC-H. A Live/Dead staining, in this case SYTOX® blue, is used to eliminate dead cells. In the final panel, the live, SYTOX® blue-negative, cells can now be analyzed for phagocytosis by plotting Cell Tracker Green CMFDA (target cells) vs. CD11c-APC (BMDCs).

    Table 1. Compensation matrix generated in FlowJo for the analysis of the phagocytic BMDCs

Data analysis

  1. Software
    1. BD FACSuiteTM software (BD)
    2. FlowJo software version 10.0.8 or newer (FlowJo)
  2. The experiment should be repeated a minimum of three times, each time including technical triplicates of each sample. The statistical difference between BMDC uptake of live cells versus that of necroptotic cells can be analyzed with a Mann-Whitney non-parametric t-test.
  3. The phagocytic BMDCs (see gating strategy in the legend of Figure 1) are calculated as the percentage of double-positive CMFDA+ CD11c+ cells (BMDCs that have engulfed target cells) out of all CD11c+ cells (the total BMDC population).

Notes

To verify the efficiency of the cell death induction in the target cells, it is advised to run a cell death analysis on the flow cytometer in parallel with the phagocytosis analysis. Cell death analysis should include the nucleic acid staining combined with an Annexin V fluorescent probe (Aaes et al., 2016). Tet-On induction for 18 h with doxycycline results in > 85% cell death.

Recipes

  1. DMEM CT26 culture medium
    Note: Store at 4 °C; heat to 37 °C before use.
    DMEM
    10% FBS
    1.3% Na-pyruvate
    1.4 mM L-glutamine
  2. RPMI BMDC/Co-culture medium
    Note: Store at 4 °C; heat to 37 °C before use.
    RPMI
    5% FBS
    1.3% Na-pyruvate
    1.4 mM L-glutamine
    50 µM 2-Me
    20 ng/ml mGM-CSF
  3. FACS buffer
    Note: Store at 4 °C.
    DPBS
    0.5% FBS
  4. Staining solution
    Note: Prepare just before use, keep on ice.
    FACS buffer
    Fc BlockTM diluted 1/250
    APC Hamster anti-mouse CD11c diluted 1/350

Acknowledgments

Peter Vandenabeele’s research group is part of the Cancer Research Institute Ghent (CRIG). Funding is supported by Interuniversity Attraction Poles (IAP 7/32), Research Foundation Flanders (FWO), Methusalem grants, Ghent University grants and the Belgian Foundation Against Cancer.
The authors declare no conflicts of interest.

References

  1. Aaes, T. L., Kaczmarek, A., Delvaeye, T., De Craene, B., De Koker, S., Heyndrickx, L., Delrue, I., Taminau, J., Wiernicki, B., De Groote, P., Garg, A. D., Leybaert, L., Grooten, J., Bertrand, M. J., Agostinis, P., Berx, G., Declercq, W., Vandenabeele, P. and Krysko, D. V. (2016). Vaccination with necroptotic cancer cells induces efficient anti-tumor immunity. Cell Rep 15(2): 274-287.
  2. Brouckaert, G., Kalai, M., Krysko, D. V., Saelens, X., Vercammen, D., Ndlovu, M. N., Haegeman, G., D'Herde, K. and Vandenabeele, P. (2004). Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production. Mol Biol Cell 15(3): 1089-1100.
  3. Krysko, D. V., Denecker, G., Festjens, N., Gabriels, S., Parthoens, E., D'Herde, K. and Vandenabeele, P. (2006). Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells. Cell Death Differ 13(12): 2011-2022.
  4. Obeid, M., Tesniere, A., Ghiringhelli, F., Fimia, G. M., Apetoh, L., Perfettini, J. L., Castedo, M., Mignot, G., Panaretakis, T., Casares, N., Metivier, D., Larochette, N., van Endert, P., Ciccosanti, F., Piacentini, M., Zitvogel, L. and Kroemer, G. (2007). Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 13(1): 54-61.

简介

该方案是基于流式细胞术的方法,以测量骨髓来源的树突细胞(BMDCs)在体外的坏死性靶细胞的吞噬效率(Aaes等人 2016)。该方法是先前发表的论文中使用的方案的稍微修改和更新的版本(Krysko等人,2006; Brouckaert等人,2004)。简言之,在细胞被诱导经历细胞死亡之前,用CellTracker TM TM染料标记靶细胞。在用BMDCs共培养2小时后,用树突细胞标记物和死细胞标记物对细胞进行免疫染色,并使用流式细胞仪定量吞噬效率。该方案可以容易地用于经历除坏死作用以外的细胞死亡模式的靶细胞。

[背景] 研究BMDCs吞噬细胞摄取的坏死细胞是检测免疫原性细胞死亡模型的初步步骤(Obeid等人,2007)。有效摄取将允许吞噬细胞将抗原交叉呈递到白细胞,从而产生针对死的靶细胞的免疫反应。在该协议中,我们使用CellTracker TM sup TM染料。这种类型的染料在某些浓度下可能是有毒的,其可以根据使用的细胞类型而变化。因此,我们建议用户首先为使用的靶细胞找到最佳的染料浓度。最佳地,CellTracker TM 染料本身不应该诱导任何细胞死亡,而是应当标记靶细胞,使得它们容易与CD11c阳性BMDC分离。

材料和试剂

  1. Delta处理的10cm培养皿(Thermo Fisher Scientific,Nunclon ,目录号:153066)
  2. 15ml聚苯乙烯离心管(Corning,Falcon ,目录号:352095)
  3. 6孔悬浮板(SARSTEDT,目录号:83.3920.500)
  4. 96 V孔,2.0ml聚丙烯板(Greiner Bio One,MASTERBLOCK ,目录号:780285)
  5. 5ml圆底聚苯乙烯管(Corning,Falcon ,目录号:352054)
  6. 从BALB/c WT小鼠分离的骨髓来源的树突细胞(BMDC)(参见程序)
  7. CT26坏死可诱导的细胞(DD_RIPK3)(Aaes等人,2016)
  8. Dulbecco改良的Eagle培养基(DMEM),高葡萄糖(Thermo Fisher Scientific,Gibco TM ,目录号:41965062)
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270106)
  10. 丙酮酸钠(Sigma-Aldrich,目录号:S8636)
  11. L-谷氨酰胺(Lonza,目录号:BE17-605F)
  12. Roswell公园纪念学院(RPMI)1640培养基(Thermo Fisher Scientific,Gibco TM ,目录号:52400025)
  13. 2-巯基乙醇(50mM)(Thermo Fisher Scientific,Gibco TM ,目录号:31350010)
  14. 小鼠GM-CSF(VIB蛋白质服务设施,UGent-VIB炎症研究中心)
  15. ACK裂解缓冲液(Lonza,目录号:10-548E)
  16. Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM ,目录号:14190094)
  17. CellTracker 绿色CMFDA染料(Thermo Fisher Scientific,Molecular Probes TM ,目录号:C7025)
  18. 多西环素(Sigma-Aldrich,目录号:D9891)
  19. B/B同源二聚体(Takara Bio,目录号:635059)
  20. 将纯化的大鼠抗小鼠CD16/CD32(小鼠BD Fc Block TM)(BD,Pharmingen TM ,目录号:553142)
  21. APC仓鼠抗小鼠CD11c(BD,Pharmingen TM ,目录号:550261)
  22. SYTOX蓝核酸染料(Thermo Fisher Scientific,Molecular Probes TM ,目录号:S11348)
  23. BD FACSuite CS& T Research Beads Kit(BD,FACSuite TM ,目录号:650621)
  24. DMEM CT26培养基(参见配方)
  25. RPMI BMDC /共培养培养基(参见配方)
  26. FACS缓冲区(参见配方)
  27. 染色溶液(见配方)

设备

  1. 台式离心机
  2. BD FACSVerse TM流式细胞仪(BD,型号:BD FACSVERSE)

软件

  1. BD FACSuite TM 软件(BD)
  2. FlowJo软件版本10.0.8或更高版本(FlowJo)

程序

通过粉碎7周龄BALB/c WT小鼠的股骨和胫骨来分离骨髓细胞。将红细胞用ACK裂解缓冲液裂解,并使用RPMI培养基 - 每2ml每6孔400,000个细胞将细胞分化为树突状细胞8天。在第三天,加入2ml新鲜培养基,并在第6天用新鲜培养基替换培养基。

  1. 靶细胞死亡诱导,CT26 DD_RIPK3,细胞
    1. 种子1×10 6个CT26 DD_RIPK3细胞在每10cm培养皿的10ml DMEM培养基中。
    2. 等待5小时细胞附着。
    3. 用无血清DMEM洗涤培养皿。
    4. 向每个板中加入10ml无血清DMEM +1μMCellTracker TM 绿色CMFDA。
    5. 在37℃孵育30分钟。
    6. 取出培养基,并用RPMI培养基洗涤平板
    7. 向每个培养皿中加入10ml RPMI培养基,在37℃下孵育2小时
    8. 通过加入1μg/ml多西环素+ 10nM B/B同型二聚体诱导细胞死亡
    9. 在37℃下再孵育18小时。

  2. 与BMDCs共培养
    1. 将BMDC和CT26靶细胞收集到单独的15ml Falcon管中
      1. 通过加入EDTA(2ml/75cm 2),在37℃下孵育2分钟,然后在RPMI培养基(每75cm 2中8ml)中收集活的CT26细胞,/sup>)。以300xg离心5分钟。
      2. 通过仅吸取溶液中的细胞来收集死的CT26细胞。以400xg离心5分钟。
    2. 计数细胞并将其重悬于RPMI培养基中
    3. 将细胞接种在6孔悬浮板中
    4. 种子400,000个BMDCs + 400,000个CT26细胞(比例1:1),总体积为4ml /孔。
    5. 在37℃下孵育2小时。
    6. 包括用于单染色和补偿控制单元的孔:
      1. CT26细胞                           ;                  未染色的目标单元格控制
      2. CT26细胞 - 细胞死亡诱导的SYTOX ?蓝色单染色控制
      3. CT26细胞                           ;                      CellTracker TM />
      4. BMDCs                                 无染色BMDC控制< br />
      5. BMDCs                                     CD11c-APC单染色控制

  3. 免疫染色
    1. 将细胞转移到深的96孔板中。
    2. 在4℃下以400xg 旋转5分钟。
    3. 通过轻拂板弃去上清液,并将沉淀物重悬在200μlFACS缓冲液中
    4. 在4℃下以400xg 旋转5分钟。
    5. 将样品重悬于200μl染色溶液(或单染剂溶液)中,每8×10 5个细胞的最终抗体浓度(APC-CD11c)为0.1μg。
    6. 在4℃下孵育30分钟,避光
    7. 洗涤样品并在4℃下以400×g离心5分钟
    8. 重悬在FACS缓冲液+1.25μMSYTOX?蓝色(或仅用于单染色样品的FACS缓冲液)中。
    9. 转移到5ml Falcon 圆底管
    10. 将样品保存在冰上(最多1小时),直到在流式细胞仪上采集
    11. 在流式细胞仪上使用FACSuite TM 获取每个样品最少50-100,000个事件。

  4. FlowiO中的门控策略
    使用允许仅分析单个细胞的门控策略来确定BMDC对CMFDA标记的死细胞材料的真实吸收,并且确定为CD11c + CMFDA + sup/+双重阳性细胞表示为所有CD11c + 细胞的百分比。有关FlowJo中使用的门控策略和补偿矩阵,请参见图1和表1

    图1.在FlowJo中用于分析的门控策略。图1显示了在FlowJo中分析结果时使用的门控策略。使用前向和侧向散射区(FSC-A对SSC-A),碎片被排除在分析之外。在以下两个步骤中,通过围绕单个细胞绘制栅极,去除双峰细胞,其大致沿着FSC-A对比FSC-H的对角线聚集,随后是SSC-A对比SSC-H。活/死染色,在这种情况下SYTOX 蓝,用于消除死细胞。在最后一组中,通过绘制细胞跟踪器绿色CMFDA(靶细胞)对CD11c-APC(BMDC),可以分析活的,SYTOX 蓝阴性细胞的吞噬作用。 >
    表1.在FlowJo中生成的用于分析吞噬BMDC的补偿矩阵

数据分析

  1. 软件
    1. BD FACSuite TM 软件(BD)
    2. FlowJo软件版本10.0.8或更高版本(FlowJo)
  2. 实验应重复至少三次,每次包括每个样品的三次重复。 BMDC对活细胞的摄取与坏死细胞的摄取之间的统计学差异可以用Mann-Whitney非参数性测试来分析。
  3. 吞噬BMDC(参见图1的图例中的门控策略)计算为双阳性CMFDA + CD11c +细胞(已经吞噬靶细胞的BMDC)的百分比,在所有CD11c + 细胞(总BMDC群体)中

笔记

为了验证靶细胞中细胞死亡诱导的效率,建议在流式细胞仪上与吞噬作用分析并行进行细胞死亡分析。细胞死亡分析应包括与Annexin V荧光探针组合的核酸染色(Aaes等人,2016)。用多西环素诱导18小时导致> 85%细胞死亡。

食谱

  1. DMEM CT26培养基
    注意:储存在4°C;使用前加热至37°C。
    DMEM
    10%FBS
    1.3%丙酮酸钠 1.4mM L-谷氨酰胺
  2. RPMI BMDC /共培养培养基
    注意:储存在4°C;使用前加热至37°C。
    RPMI
    5%FBS
    1.3%丙酮酸钠 1.4mM L-谷氨酰胺 50μM2-Me
    20ng/ml mGM-CSF
  3. FACS缓冲区
    注意:存储在4°C。
    DPBS
    0.5%FBS
  4. 染色溶液
    注意:请在使用前准备好冰箱。
    FACS缓冲区
    Fc Block TM 稀释1/250
    APC仓鼠抗小鼠CD11c稀释1/350

致谢

Peter Vandenabeele的研究小组是根特癌症研究所(CRIG)的一部分。资助由国际大学吸引波兰学院(IAP 7/32),研究基金会佛兰德斯大学(FWO),Met耶路撒冷补助金,根特大学补助金和比利时癌症基金会支持。
作者声明没有利益冲突。

参考文献

  1. Aaes,TL,Kaczmarek,A.,Delvaeye,T.,De Craene,B.,De Koker,S.,Heyndrickx,L.,Delrue,I.,Taminau,J.,Wiernicki,B.,De Groote,P 。,Garg,AD,Leybaert,L.,Grooten,J.,Bertrand,MJ,Agostinis,P.,Berx,G.,Declercq,W.,Vandenabeele,P.and Krysko,DV(2016) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27050509"target ="_ blank">用坏死性癌细胞接种诱导有效的抗肿瘤免疫。 > Cell Rep 15(2):274-287
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aaes, T. L., Krysko, D. V. and Vandenabeele, P. (2016). Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs. Bio-protocol 6(21): e1997. DOI: 10.21769/BioProtoc.1997.
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