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
发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1091 浏览次数: 13382
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
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
- 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
- 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 )
- RPMI 1640 with L-glutamine (Lonza, catalog number: BE12-702F ) containing 10% FBS with 100 U/100 μg/ml penicillin/streptomycin
- FBS (Biochrom, catalog number: S0615 )
- Dulbecco’s Phosphate Buffered Saline (DPBS) (Biochrom, catalog number: L1825 )
- 1x 0.05% Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300 )
- Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco®, catalog number: 14025 ) containing 6 mM glucose (starvation medium)
- Rapamycin from Streptomyces hygroscopicus (1-5 µmol/L) (Sigma-Aldrich, catalog number: R0395 )
- PP242 hydrate (1-5 µmol/L) (Sigma-Aldrich, catalog number: P0037 )
- 3-methyladenine (3-MA) (3-10 mmol/L) (Sigma-Aldrich, catalog number: M9281 )
- Wortmannin (30-100 nmol/L) (Sigma-Aldrich, catalog number: W3144 )
- LY294002 (7-20 µmol/L) (Sigma-Aldrich, catalog number: L9908 )
- Nocodazole (12-50 µmol/L) (Sigma-Aldrich, catalog number: M1404 )
- Vinblastine (12-50 µmol/L) (Sigma-Aldrich, catalog number: V1377 )
- Ammonium chloride (NH4Cl) (10-20 mmol/L) (Sigma-Aldrich, catalog number: A0171 )
- Hydrohychloroquine sulphate (HCQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: H0915 )
- Chloroquine (CQ) (5-10 µmol/L) (Sigma-Aldrich, catalog number: C6628 )
- Dimethyl sulfoxide DMSO (Sigma-Aldrich, catalog number: D8418 )
Equipment
- 37 °C, 5% CO2 humidified incubator
- Centrifuge
- FACSCalibur, LSR II (BD) or analogous equipment
Procedure
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. 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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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
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