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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

MS Metodi Stankov
Diana Panayotova-Dimitrova Diana Panayotova-Dimitrova
ML Martin Leverkus
Jan-Henning Klusmann Jan-Henning Klusmann
Georg Behrens Georg Behrens

发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1090 浏览次数: 27551

评审: Anonymous reviewer(s)

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

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

English
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文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

微生物学 > 抗微生物试验 > 自体吞噬试验

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

细胞生物学 > 细胞成像 > 活细胞成像

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