Flow cytometry based assay for autophagic flux

In 2010, we published a flow-cytometry based assay for measuring autophagic flux (Eng et al., 2010). The assay is simple and reliable and can be used to quantify autophagic flux in many types of cells, with or without previous transfection with the EGFP-LC3 reporter construct.

Saponin extraction allows quantification of EGFP-LC3-II fluorescence by FACS.

(A) HOS-EGFP-LC3 cells were starved of amino acids or chloroquine (CQ) treated for 4 hours or left untreated. EGFP fluorescence was measured by FACS either without (top) or with (bottom) a wash with PBS containing 0.05% saponin.

(B) Schematic diagram of the effects of the saponin wash. Due to the reorganization of the EGFP-LC3 reporter protein, induction of autophagosome formation should not change the total levels of fluorescence in HOS-EGFP-LC3 expressing cells. However, extraction of EGFPLC3-I with saponin would be expected to result in a higher level of fluorescence in cells with proportionally higher levels of EGFP-LC3-II-containing autophagosomes.

Protocol

Beforehand:

1. Seed cells in 6-well plates

2. Include a positive control, chloroquine at 50 uM

Protocol:

Trypsinisation and saponin rinse:

1. Add 0.5 ml trypsin to each well, incubate 37°C until cells have detached

2. Add 3ml medium to each well and transfer to FACS tube

3. Spin 5min, 1600 rpm

4. Discard supernatant and resuspend in 3ml PBS

5. Spin 5min, 1600 rpm

6. Discard supernatant and resuspend in 0.5ml of 0.05% saponin (diluted in PBS)

7. Vortex briefly

8. Add 3ml PBS per tube and spin 5min, 1600 rpm

9. Discard supernatant by tipping the liquid off (there should still be a small amount of liquid left in the tube, around 50ul)

10. FACS

Intracellular staining:

10. Dilute primary antibody (mouse anti LC3, MBL M152-3) 1:20 in 0.05% saponin

11. Add 1ul of the 1:20 dilution, to each tube. Vortex

12. Incubate 20 min at room temp

13. Add 3ml PBS per tube, spin 5min 1600 rpm

14. Discard supernatant by tipping liquid off (should still have small amount of liquid left in tube)

15. Dilute secondary antibody (goat anti mouse IgG-RPE, Southern Biotech 1030-09S) 1:20 in 0.05% saponin

16. Add 1ul of the 1:20 dilution, to each tube. Vortex

17. Incubate 20 min, room temp, dark

18. Add 3ml PBS per tube, spin, discard sup

19. Repeat PBS rinse

20. FACS

Publications describing this assay

Klionsky, D. et al., approx 1200 authors listed alphabetically, including Eng, K, and McInerney, G.M. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 8:445-544.

Eng, K.E., Panas, M.D., Murphy, D., Karlsson Hedestam, G.B. and McInerney, G.M. (2012). Accumulation of autophagosomes in Semliki Forest virus infected cells is dependent on the expression of viral structural proteins. J Virol 86:5674-5685.

Eng, K.E., Panas, M., Karlsson Hedestam, G.B., and McInerney, G.M. (2010). A novel flow cytometry based assay for autophagic flux. Autophagy 6:634-641.