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Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells
使用Cyto-ID染色原代细胞并用流式细胞法分析其自噬活性   

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Abstract

Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the autophagosomal content might be. The method is based on Cyto-ID staining of autophagic compartments (pre-autophagosomes, autophagosomes, and autophagolysosomes) in live cells using Cyto-ID® Autophagy Detection Kit. Autophagic compartments are intermediate constituents of a dynamic lysosomal degradation process and their intracellular abundance at a particular time point is a function of the established equilibrium between their generation and degradation. Determination of autophagic flux facilitates the discrimination between early induction of autophagosome formation and late inhibition of autophagosome maturation as both results in an ultimate increase in autophagosomal presence. Cyto-ID assay is based on the usage of a specific dye that selectively stains autophagic compartments and therefore allows determination of autophagic flux as accumulation of stained compartments in basic or activated conditions [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L) or Hanks’ Balanced Salt Solution containing 6 mmol/L glucose (starvation medium)] after blockage of autophagolysosomal degradation using lysosomotropic compounds such as ammonium chloride (NH4Cl) (10-20 mmol/L) or chloroquine (CQ) (5-10 µmol/L). ΔMFI Cyto-ID = MFI Cyto-ID (+CQ/NH4Cl) - MFI Cyto-ID (-CQ/NH4Cl).

Keywords: Autophagy(自噬), Primary Cells(原代细胞), Cyto-ID(细胞的身份), Autophagic flux(自噬通量), Flow Cytometry(流式细胞仪)

Materials and Reagents

  1. Cells lines of interest (HepG2, HUH7, CMK, K562 etc.) or primary cells [for example: murine bone marrow-derived dendritic cells (BMDCs)]
  2. Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences, catalog number: ENZ-51031-K200 )
  3. Eagle's minimal essential medium (EMEM) (ATCC, catalog number: 30-2003 ) containing 10% fetal bovine serum (FBS) (Biochrom, catalog number: S0615 ) with 100 U/100 μg/ml penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  4. RPMI 1640 with L-glutamine (Lonza, catalog number: BE12-702F ) containing 10% FBS with 100 U/100 μg/ml penicillin/streptomycin
  5. DMEM (low glucose, pyruvate, no glutamine, no phenol red) (Life Technologies, Gibco®, catalog number: 11880-028 )
  6. Dulbecco’s Phosphate Buffered Saline (DPBS) (Biochrom, catalog number: L1825 )
  7. 1x 0.05% Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300 )
  8. Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco®, catalog number: 14025 ) containing 6 mM glucose (starvation medium)
  9. Rapamycin from Streptomyces hygroscopicus (1-5 µmol/L) (Sigma-Aldrich, catalog number: R0395 )
  10. PP242 hydrate (1-5 µmol/L) (Sigma-Aldrich, catalog number: P0037 )
  11. 3-methyladenine (3-MA) (3-10 mmol/L) (Sigma-Aldrich, catalog number: M9281 )
  12. Wortmannin (30-100 nmol/L) (Sigma-Aldrich, catalog number: W3144 )
  13. LY294002 (7-20 µmol/L) (Sigma-Aldrich, catalog number: L9908 )
  14. Nocodazole (12-50 µmol/L) (Sigma-Aldrich, catalog number: M1404 )
  15. Vinblastine (12-50 µmol/L) (Sigma-Aldrich, catalog number: V1377 )
  16. Ammonium chloride (NH4Cl) (10-20 mmol/L) (Sigma-Aldrich, catalog number: A0171 )
  17. Hydrohychloroquine sulphate (HCQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: H0915 )
  18. Chloroquine (CQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: C6628 )
  19. Dimethyl sulfoxide DMSO (Sigma-Aldrich, catalog number: D8418 )

Equipment

  1. 37 °C, 5% CO2 humidified incubator
  2. Centrifuge
  3. FACSCalibur, LSR II (BD) or analogous equipment

Procedure

  1. Maintain cells under standard tissue culture conditions at 37 °C, 5% CO2 in a humidified incubator. Keep cell density below 1 x 106/ml.
    Caution: Prior to analysis cell should be kept for several hours (min 12 h) in fresh medium to avoid potential activation of autophagy due to nutrients exhaustion. Generally culture medium contains autophagy affecting substances: amino acids, glucose, growth factors, hormones etc. Take care when comparing autophagic flux in different conditions to normalize for all the necessary factors. Normalize also for the solvent used when analysing the effect of different substances on autophagy – for example DMSO, ethanol etc. might affect autopagic flux.
  2. Incubate the cells for the desired time and under the conditions of interest. Negative control subjected to vehicle treatment should be included.
    Caution: When analysing prolonged periods of time and/or under conditions potentially affecting cells numbers or viability, differences in nutrient consumption and therefore abundance might occur and influence your results as autophagic activity is highly related to the nutritional status.
  3. Positive control [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L), Hanks’ Balanced Salt Solution containing 6 mmol/L glucose (starvation medium)] and negative control [3-methyladenine (3-MA) (3-10 mmol/L), wortmannin (30-100 nmol/L), LY294002 (7-20 µmol/L), nocodazole (12-50 µmol/L), vinblastine (12-50 µmol/L), ammonium chloride (NH4Cl) (10-20 mmol/L), hydrohychloroquine (HCQ) or chloroquine (CQ) (5-10 µmol/L)] may be also included.
  4. Upon completion of treatment period, wash the cells with DPBS. For suspension cells use centrifugation, for adherent cells wash as they are adherent (do not trypsinize).
  5. Resuspend suspension cells, or add to adherent cells Cyto-ID Green containing indicator free cell culture medium, containing 5% FBS. Suggested cell density for staining stage is 105 to 106 cells/ml. Suggested Cyto-ID Green concentration is 1 μl of Cyto-ID Green Detection Reagent in 1 ml cell culture medium.
  6. Mix well and incubate for 30 min under standard tissue culture conditions at 37 ºC, 5% CO2 in the dark.
  7. At the end of staining procedure, wash away the Cyto-ID containing medium. Wash once more with DPBS and resuspend suspension cells or trypsinize, wash and resuspend adherent cells in ice cold DPBS containing 2% FBS.
    Note: Caution upon completion of staining step whenever possible keep always on ice and in the dark.
  8. Analyze samples using flow cytometer and plot data of cell counts as FITC (FL1) fluorescence intensity. Table 1 may be used to facilitate the interpretation of the results.

    Table 1. Analysis of unknown substance X
     X plus   
    Medium    
    Activator
    I   
    →   
    ↑↑
    II   
    ↑↑   
    ↑↑↑
    III   
    ↓↓   
    ↓ or ↑

    Notes:
    1. →↑↓: ΔMFI Cyto-ID = MFI Cyto-ID (+CQ/NH4Cl) - MFI Cyto-ID (-CQ/NH4Cl).
      CQ/NH4Cl stays for either CQ or NH4Cl.
      Caution: The calculation of ΔMFI Cyto-ID requires both +CQ/NH4Cl and -CQ/NH4Cl condition for each individual sample!
    2. Suggested incubation with ammonium chloride (NH4Cl) (10–20 mmol/L) or chloroquine (CQ) (5–10 µmol/L) is 5 h.
    3. (Activator): autophagy induction [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L) or starvation medium]. Suggested incubation 5 h. (Caution: The concentration of the activator should be established for the particular cell type.)
    4. Analysis of unknown substance X should include co incubation with one or more activators. Table 1 illustrates three possible scenarios. a) Scenario (I) unknown substance X does not impact cellular autophagic activity; b) Scenario (II) X is an activator; c) Scenario (III) X is an inhibitor of autophagy.
    5. Caution: Although considered the most accurate sensitive and reliable method for analysis of autophygic flux, flow cytometry determination of Cyto-ID stained cells should be combined with alternative methods with non overlapping limitations such as electron microscopy (EM) fluorescence microscopy (FM), western blotting (WB) etc.
    6. Example data:


      Figure 1. Flow cytometry analysis of autophagic flux in BMDC incubated with autophagy inhibitor 3MA (10 mM) for 10 h in the presence or absence of autophagy activator PP242 for the last 5 h (left) with representative histograms (right)


Acknowledgments

Stankov, M and Behrens, G were supported by the DFG (BE-2089/2-1 and EXC62/1). Klusmann, J was supported by a grant from the Wilhelm Sander-Foundation (2011.057.1), the German Research Foundation (DFG; KL-2374/1-1). Klusmann, J is a fellow of the Emmy Noether-Programme from the DFG (KL-2374/2-1). Leverkus, M by the DFG (LE-953/8-1 and LE-953/6-1) and the Mildred-Scheel-Stiftung (#109891).

References

  1. Cyto-ID® Autophagy Detection Kit Manual (Enzo Life Sciences). http://www.enzolifesciences.com/fileadmin/enzo/Manuals/51031-K200.pdf.
  2. Mizushima, N., Yoshimori, T. and Levine, B. (2010). Methods in mammalian autophagy research. Cell 140(3): 313-326.
  3. Sinicrope, F. A., Sirko, A., Siu, P. M. et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8(4): 445-544. 
  4. Stankov, M. V., El Khatib, M., Kumar Thakur, B., Heitmann, K., Panayotova-Dimitrova, D., Schoening, J., Bourquin, J. P., Schweitzer, N., Leverkus, M., Welte, K., Reinhardt, D., Li, Z., Orkin, S. H., Behrens, G. M. and Klusmann, J. H. (2014). Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy. Leukemia 28(3): 577-588. 

简介

流式细胞术允许非常灵敏和可靠的高通量分析自噬通量。该方法允许在流动中筛选细胞并捕获多组分图像。使用这种技术,可以在悬浮液以及粘附细胞中在胰蛋白酶消化时精确分析自噬流,而与自噬体内容物的异质性无关。该方法基于使用Cyto-ID自动吞噬检测试剂盒的活细胞中的自体吞噬细胞(前自噬体,自噬体和自噬溶酶体)的Cyto-ID染色。自噬区室是动态溶酶体降解过程的中间组分,并且它们在特定时间点的细胞内丰度是其产生和降解之间建立的平衡的函数。自噬吞噬的确定有助于自噬体形成的早期诱导和自噬体成熟的晚期抑制之间的区分,因为两者都导致自噬体存在的最终增加。 Cyto-ID测定基于使用选择性染色自噬隔室的特定染料,因此允许测定自噬通量,作为染色隔室在碱性或活化条件[雷帕霉素(1-5μmol/L),PP242(1- (NH 4 Cl)(10-20μmol/L)或含有6mmol/L葡萄糖(饥饿培养基)的Hanks平衡盐溶液),使用溶酶体化合物阻断自噬溶酶体降解mmol/L)或氯喹(CQ)(5-10μmol/L)。 ΔMFICyto-ID = MFI Cyto-ID(+ CQ/NH 4 Cl)-MFI Cyto-ID(-CQ/NH 4 Cl)。

关键字:自噬, 原代细胞, 细胞的身份, 自噬通量, 流式细胞仪

材料和试剂

  1. 感兴趣的细胞系(HepG2,HUH7,CMK,K562等)或原代细胞[例如:鼠骨髓衍生的树突细胞(BMDC)]
  2. Cyto-ID 自噬检测试剂盒(Enzo Life Sciences,目录号:ENZ-51031-K200)
  3. 含有100U /100μg/ml青霉素/链霉素(Life Technologies,Gibco)的含有10%胎牛血清(FBS)(Biochrom,目录号:S0615)的Eagle's最小必需培养基(EMEM)(ATCC,目录号:30-2003) ®,目录号:15140-122)
  4. 含有10%FBS和100U /100μg/ml青霉素/链霉素的含有L-谷氨酰胺的RPMI 1640(Lonza,目录号:BE12-702F)
  5. DMEM(低葡萄糖,丙酮酸盐,无谷氨酰胺,无酚红)(Life Technologies,Gibco ,目录号:11880-028)
  6. Dulbecco's磷酸盐缓冲盐水(DPBS)(Biochrom,目录号:L1825)
  7. 1x 0.05%胰蛋白酶-EDTA(酚红)(Life Technologies,目录号:25300)
  8. 含有6mM葡萄糖(饥饿培养基)的Hanks平衡盐溶液(HBSS)(Life Technologies,Gibco ,目录号:14025)
  9. 来自吸湿链霉菌(1-5μmol/L)(Sigma-Aldrich,目录号:R0395)的雷帕霉素
  10. PP242水合物(1-5μmol/L)(Sigma-Aldrich,目录号:P0037)
  11. 3-甲基腺嘌呤(3-MA)(3-10mmol/L)(Sigma-Aldrich,目录号:M9281)
  12. 渥曼青霉素(30-100nmol/L)(Sigma-Aldrich,目录号:W3144)
  13. LY294002(7-20μmol/L)(Sigma-Aldrich,目录号:L9908)
  14. 诺可达唑(12-50μmol/L)(Sigma-Aldrich,目录号:M1404)
  15. 长春花碱(12-50μmol/L)(Sigma-Aldrich,目录号:V1377)
  16. 氯化铵(NH 4 Cl)(10-20mmol/L)(Sigma-Aldrich,目录号:A0171)
  17. 硫酸氢氯喹(HCQ)(5-10μmol/L)(Sigma-Aldrich,目录号:H0915)
  18. 氯喹(CQ)(5-10μmol/L)(Sigma-Aldrich,目录号:C6628)
  19. 二甲基亚砜DMSO(Sigma-Aldrich,目录号:D8418)

设备

  1. 37℃,5%CO 2湿润培养箱
  2. 离心机
  3. FACSCalibur,LSR II(BD)或类似设备

程序

  1. 维持细胞在标准组织培养条件下在37℃,5%CO 2在湿润的培养箱中。保持细胞密度低于1×10 6 /μL/ml。
    注意:在分析之前,细胞应在新鲜培养基中保持几小时(最少12小时),以避免由于营养物耗尽而导致潜在的自噬激活。一般培养基包含自噬影响物质:氨基酸,葡萄糖,生长因子,激素等。在比较不同条件下的自噬通量时要注意所有必要因素的正常化。在分析不同物质对自噬的影响时,例如DMSO,乙醇等,也可以影响自动通量。
  2. 在所需的时间和在感兴趣的条件下孵育细胞。应包括经过载体处理的阴性对照。
    注意:当分析延长的时间段和/或潜在地影响细胞数量或存活力的条件下时,营养物消耗和因此丰度的差异可能发生,并且影响自我结果,因为自噬活性与营养状态高度相关。
  3. 阳性对照[雷帕霉素(1-5μmol/L),PP242(1-5μmol/L),含6mmol/L葡萄糖的Hanks平衡盐溶液(饥饿培养基)]和阴性对照[3-甲基腺嘌呤),枸杞素(30-100nmol/L),LY294002(7-20μmol/L),诺考达唑(12-50μmol/L),长春花碱还可以包括氯化铵(NH 4 Cl)(10-20mmol/L),氢氯喹(HCQ)或氯喹(CQ)(5-10μmol/L)]。
  4. 治疗期结束后,用DPBS清洗细胞。对于悬浮细胞使用离心,对于粘附细胞,因为它们是粘附的(不胰蛋白酶消化),所以洗涤
  5. 重悬悬浮细胞,或添加到贴壁细胞Cyto-ID Green含有指示剂的细胞培养基,含有5%FBS。染色阶段的建议细胞密度为10 5至10 6个细胞/ml。建议的Cyto-ID绿色浓度是1ml Cyto-ID绿色检测试剂在1ml细胞培养基中。
  6. 充分混合并在标准组织培养条件下在37℃,5%CO 2在黑暗中孵育30分钟。
  7. 在染色程序结束时,洗去含Cyto-ID的培养基。再次用DPBS洗涤并重悬浮悬浮细胞或胰蛋白酶消化,洗涤并将贴壁细胞悬浮在含有2%FBS的冰冷的DPBS中。
    注意:在染色步骤完成后注意,尽可能保持在冰上和在黑暗中。
  8. 使用流式细胞仪分析样品和细胞计数的绘图数据作为FITC(FL1)荧光强度。表1可用于方便解释结果。

    表1.未知物质X的分析
      X plus   
    中    
    激活
    I   
    →   
    ↑↑
    II   
    ↑↑   
    ↑↑↑
    III   
    ↓↓   
    ↓或↑

    注意:
    1. →↑↓:ΔMFICyto-ID = MFI Cyto-ID(+ CQ/NH (-CQ/NH 4 Cl)。 CQ/NH4Cl留在CQ或NH4Cl。
      警告:ΔMFICyto-ID的计算需要+ CQ/NH 4 Cl和-CQ/NH
    2. 建议与氯化铵(NH 4 Cl)(10-20mmol/L)或氯喹(CQ)(5- 10μmol/L)为5小时。
    3. (活化剂):自噬诱导[雷帕霉素(1-5μmol/L),PP242(1-5μmol/L)或饥饿培养基]。建议温育5 h。 (注意:应针对特定细胞类型确定活化剂的浓度。)
    4. 未知物质X的分析应包括与一种或多种激活剂的共孵育。表1示出了三种可能的情况。 a)情况(I)未知物质X不影响细胞自噬活性; b)情况(II)X是活化剂; c)情况(III)X是自噬的抑制剂。
    5. 注意:虽然被认为是用于分析自溶通量的最准确的灵敏和可靠的方法,但是Cyto-ID染色细胞的流式细胞术测定应当与具有非重叠限制的替代方法组合,例如电子显微镜(EM)荧光显微镜(FM ),western印迹(WB)等。
    6. 示例数据:


      图1.在自噬抑制剂3MA(10mM)与自噬激活物PP242存在或不存在的情况下在最后5小时(左)中与自噬抑制剂3MA(10mM)孵育的BMDC中的自噬通量的流式细胞术分析,/strong>


致谢

Stankov,M和Behrens,G由DFG(BE-2089/2-1和EXC62/1)支持。 Klusmann,J获得了Wilhelm Sander-Foundation(2011.057.1),德国研究基金会(DFG; KL-2374/1-1)的资助。 Klusmann,J是DFG(KL-2374/2-1)的Emmy Noether程序的研究员。 Leverkus,M由DFG(LE-953/8-1和LE-953/6-1)和Mildred-Scheel-Stiftung(#109891)。

参考文献

  1. Cyto-ID ®自噬检测试剂盒手册(Enzo Life Sciences)。 http://www.enzolifesciences.com/fileadmin/enzo/Manuals/51031 -K200.pdf。
  2. Mizushima,N.,Yoshimori,T。和Levine,B。(2010)。 哺乳动物自噬研究中的方法。细胞 140(3 ):313-326。
  3. Sinicrope,F.A.,Sirko,A.,Siu,P.M.et al。(2012)。 监测自噬的测定法的使用和解释指南。 Autophagy 8(4):445-544。
  4. Stankov,MV,El Khatib,M.,Kumar Thakur,B.,Heitmann,K.,Panayotova-Dimitrova,D.,Schoening,J.,Bourquin,JP,Schweitzer,N.,Leverkus,M.,Welte,K 。,Reinhardt,D.,Li,Z.,Orkin,SH,Behrens,GM和Klusmann,JH(2014)。 组蛋白脱乙酰酶抑制剂通过抑制自噬而诱导骨髓性白血病的凋亡。白血病 28(3):577-588。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Stankov, M., Panayotova-Dimitrova, D., Leverkus, M., Klusmann, J. and Behrens, G. (2014). Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells. Bio-protocol 4(7): e1090. DOI: 10.21769/BioProtoc.1090.
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