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Flow Cytometric Analyses of Autophagic Activity using LC3-GFP fluorescence
采用LC3B-GFP荧光对自噬活性进行流式细胞检测分析   

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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 LC3 punctae content might be. The method is based on the fact that intra-cellularly expressed LC3-GFP serves as a potential autophagic substrate for degradation. Therefore changes in total intracellular LC3-GFP fluorescence intensity is used as an indicator of cellular autophagic activity in living cells. Increased autophagic flux is expected to result in a progressive delivery of LC3-GFP to autolysosome where this substrate undergoes degradation. Therefore, enhanced autophagic flux is detected as a decreased total cellular GFP signal. On the other hand an inhibition of autophagic flux independent of the stage (autophagosome formation, maturation or acidification) leads to accumulation of undegraded LC3-GFP and may be detected as an enhanced intracellular GFP signal. (Caution: This methodology is based on the assumption that LC3-GFP is expressed constitutively by the model system. Data from analysis of substances or conditions influencing cellular LC3-GFP expression should be interpreted with care.)

Materials and Reagents

  1. Cell lines of interest (HepG2, HUH7, CMK, K562 etc.) stably expressing LC3-GFP
    We recommend the following commercially available plasmids: pBABEpuro GFP-LC3 (plasmid 22405) and pBABE-puro mCherry-EGFP-LC3B (plasmid 22418) generated by Jayanta Debnath from Addgene to be inserted into retroviral constructs and used for cell transduction
  2. Eagle's minimal essential medium (EMEM) (ATCC, catalog number: 30-2003 ) containing 10% fetal bovine serum (FBS) with 100 U/100 μg/ml penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  3. RPMI 1640 with L-glutamine (Lonza, catalog number: BE12-702F ) containing 10% FBS with 100 U/100 μg/ml penicillin/streptomycin
  4. FBS (Biochrom, catalog number: S0615 )
  5. Dulbecco’s Phosphate Buffered Saline (DPBS) (Biochrom, catalog number: L1825 )
  6. 1x 0.05% Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300 )
  7. Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco®, catalog number: 14025 ) containing 6 mM glucose (starvation medium)
  8. Rapamycin from Streptomyces hygroscopicus (1-5 µmol/L) (Sigma-Aldrich, catalog number: R0395 )
  9. PP242 hydrate (1-5 µmol/L) (Sigma-Aldrich, catalog number: P0037 )
  10. 3-methyladenine (3-MA) (3-10 mmol/L) (Sigma-Aldrich, catalog number: M9281 )
  11. Wortmannin (30-100 nmol/L) (Sigma-Aldrich, catalog number: W3144 )
  12. LY294002 (7-20 µmol/L) (Sigma-Aldrich, catalog number: L9908 )
  13. Nocodazole (12-50 µmol/L) (Sigma-Aldrich, catalog number: M1404 )
  14. Vinblastine (12-50 µmol/L) (Sigma-Aldrich, catalog number: V1377 )
  15. Ammonium chloride (NH4Cl) (10-20 mmol/L) (Sigma-Aldrich, catalog number: A0171 )
  16. Hydrohychloroquine sulphate (HCQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: H0915 )
  17. Chloroquine (CQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: C6628 )
  18. 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 the cells under standard tissue culture conditions at 37 ºC, 5% CO2 in a humidified incubator. Keep cell density below 1 x 106/ml and analyse at subconfluent stages.
    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 under 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.
    Caution: When analysing prolonged periods of time under conditions potentially affecting cell 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 controls [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L), Hanks’ Balanced Salt Solution containing 6 mmol/L glucose (starvation medium)] and negative controls [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. Positive controls activate autophagy and should result in a decreased total cellular LC3-GFP signal. Negative controls inhibit different stages of autophagic process and generally should lead to an increase in total cellular LC3-GFP signal.
    We also recommend analysing the effect of a new substance not only on basic, but also on rapamycin, PP242 or starvation activated autophagic flux.
  4. At the end of incubation period transfer the cells to FACS tubes, put them on ice and analyse by flow cytometry plotting the data of cell counts as GFP/FITC fluorescence intensity measured on FL1 channel. For adherent cells, wash once with DPBS, trypsinize, wash again with DPBS, resuspend in DPBS (2% FBS), put on ice and analyse.
    Caution: Generally pharmacological inhibitors of autophagy lack specificity and therefore some cell type specific effects might occur.
    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. →↑↓: Change in LC3-GFP mean fluorescence intensity (MFI).
    2. (Activator): autophagy induction [rapamycin (1-5 µmol/L), PP242 (1-5 µmol/L) or starvation medium]. Suggested incubation time 4 to 6 h.
    3. Analysis of unknown substance X should include co incubation with one or more activators. Table 1 illustrates three possible scenarios. a) Scenario (I) the 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.
    4. Caution: Although considered the most accurate sensitive and reliable method for analysis of autophygic flux, flow cytometry determination of LC3-GFP turnover should be combined with alternative methods with non overlapping limitations such as electron microscopy (EM) fluorescence microscopy (FM), western blotting (WB) etc.
    5. Example data:


      Figure 1. Flow cytometry analysis of autophagic flux in K562 cells expressing LC3-GFP fusion protein incubated with autophagy inhibitor 3MA (10 mM) in the presence or absence of autophagy activation through starvation (HBSS) (left) with representative histograms (right)


Acknowledgments

Stankov, M and Behrens, G were supported by the German Research Foundation (KFO 250, BE 2089/2-1 and Excellence Cluster REBIRTH, EXC 62/1). Leverkus, M was supported by the German Research Foundation (LE-953/5-1 and LE-953/6-1).

References

  1. 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.
  2. Mizushima, N., Yoshimori, T. and Levine, B. (2010). Methods in mammalian autophagy research. Cell 140(3): 313-326. 
  3. 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.
  4. Stankov, M. V., Panayotova-Dimitrova, D., Leverkus, M., Schmidt, R. E. and Behrens, G. M. (2013). Thymidine analogues suppress autophagy and adipogenesis in cultured adipocytes. Antimicrob Agents Chemother 57(1): 543-551. 

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。使用该技术,可以在悬浮液以及粘附细胞中在胰蛋白酶作用下精确分析自噬通量,而与LC3斑点含量可能是多样性无关。该方法基于以下事实:细胞内表达的LC3-GFP作为降解的潜在自噬底物。因此,总细胞内LC3-GFP荧光强度的变化用作活细胞中细胞自噬活性的指标。预期增加的自噬通量导致LC3-GFP逐渐递送至自溶酶体,其中该底物经历降解。因此,增强的自噬通量被检测为减少的总细胞GFP信号。另一方面,独立于阶段(自噬体形成,成熟或酸化)的自噬通量的抑制导致未降解的LC3-GFP的积累,并且可以被检测为增强的细胞内GFP信号。 (注意:该方法基于LC3-GFP由模型系统组成型表达的假设,应当小心地解释影响细胞LC3-GFP表达的物质或条件的分析数据。

材料和试剂

  1. 稳定表达LC3-GFP的感兴趣的细胞系(HepG2,HUH7,CMK,K562等) 我们推荐以下可商购的质粒:由来自Addgene的Jayanta Debnath产生的pBABEpuro GFP-LC3(质粒22405)和pBABE-puro mCherry-EGFP-LC3B(质粒22418)插入逆转录病毒构建体中并用于细胞转导
  2. 包含含有100U /100μg/ml青霉素/链霉素(Life Technologies,Gibco)的10%胎牛血清(FBS)的Eagle's最小必需培养基(EMEM)(ATCC,目录号:30-2003) ,目录号:15140-122)
  3. 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)
  4. FBS(Biochrom,目录号:S0615)
  5. Dulbecco's磷酸盐缓冲盐水(DPBS)(Biochrom,目录号:L1825)
  6. 1x 0.05%胰蛋白酶-EDTA(酚红)(Life Technologies,目录号:25300)
  7. 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)
  8. 来自吸湿链霉菌(1-5μmol/L)(Sigma-Aldrich,目录号:R0395)的雷帕霉素
  9. PP242水合物(1-5μmol/L)(Sigma-Aldrich,目录号:P0037)
  10. 3-甲基腺嘌呤(3-MA)(3-10mmol/L)(Sigma-Aldrich,目录号:M9281)
  11. 渥曼青霉素(30-100nmol/L)(Sigma-Aldrich,目录号:W3144)
  12. LY294002(7-20μmol/L)(Sigma-Aldrich,目录号:L9908)
  13. 诺可达唑(12-50μmol/L)(Sigma-Aldrich,目录号:M1404)
  14. 长春花碱(12-50μmol/L)(Sigma-Aldrich,目录号:V1377)
  15. 氯化铵(NH 4 Cl)(10-20mmol/L)(Sigma-Aldrich,目录号:A0171)
  16. 硫酸氢氯喹(HCQ)(5-10μmol/L)(Sigma-Aldrich,目录号:H0915)
  17. 氯喹(CQ)(5-10μmol/L)(Sigma-Aldrich,目录号:C6628)
  18. 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. 保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/sup/ml,并在亚汇合阶段进行分析。
    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小心,以便对所有必要因素进行归一化。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. 孵育细胞所需的时间和在感兴趣的条件下。
    警告:当在潜在影响细胞数量或活力的条件下分析长时间段时,营养物消耗和因此丰度的差异可能发生,并且影响您的结果,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),含有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)]。阳性对照激活自噬,应导致总细胞LC3-GFP信号减少。阴性对照抑制自噬过程的不同阶段,通常应导致总细胞LC3-GFP的增加 信号 我们还建议分析一种新物质不仅对基础的,而且对雷帕霉素,PP242或饥饿激活的自噬通量的影响。
  4. 在孵育期结束时,将细胞转移至FACS管,将其置于冰上,并通过流式细胞术分析细胞计数的数据,作为在FL1通道上测量的GFP/FITC荧光强度。对于贴壁细胞,用DPBS洗涤一次,胰蛋白酶化,再次用DPBS洗涤,重悬于DPBS(2%FBS)中,置于冰上并分析。 注意:通常自噬的药理学抑制剂缺乏特异性,因此可能发生一些细胞类型特异性效应。
    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. →↑↓:LC3-GFP平均荧光强度(MFI)的变化。
    2. (活化剂):自噬诱导[雷帕霉素(1-5μmol/L),PP242(1-5μmol/L)或饥饿培养基]。建议的孵化时间为4至6小时。
    3. 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。
    4. 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LC3-GFP周转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) ,免疫印迹(WB)等。
    5. 示例数据:


      图1.在存在或不存在通过饥饿(HBSS)的自噬激活(HBSS)(左)和自噬抑制剂3MA(10mM)孵育的表达LC3-GFP融合蛋白的K562细胞中的自噬通量的流式细胞术分析, 右)


致谢

Stankov,M和Behrens,G由德国研究基金会(KFO 250,BE 2089/2-1和Excellence Cluster REBIRTH,EXC 62/1)支持。 Leverkus,M由德国研究基金会(LE-953/5-1和LE-953/6-1)支持。

参考文献

  1. Sinicrope,F.A.,Sirko,A.,Siu,P.M.et al。 (2012)。 监测自噬的检测方法的使用和解释指南 自噬 8(4):445-544
  2. Mizushima,N.,Yoshimori,T。和Levine,B。(2010)。 哺乳动物自噬研究中的方法。 140(3):313-326。 
  3. 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。
  4. Stankov,M.V.,Panayotova-Dimitrova,D.,Leverkus,M.,Schmidt,R.E.and Behrens,G.M。(2013)。 胸苷类似物抑制培养的脂肪细胞中的自噬和脂肪形成。抗微生物剂化疗 57(1):543-551。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Stankov, M., Panayotova-Dimitrova, D., Leverkus, M. and Behrens, G. (2014). Flow Cytometric Analyses of Autophagic Activity using LC3-GFP fluorescence. Bio-protocol 4(7): e1091. DOI: 10.21769/BioProtoc.1091.
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