Measurement of Autophagic Flux

Autophagic flux was measure using the CYTO-ID autophagy detection kit (Enzo Life Sciences ENZ-51031). HCC1806 and HT29 cells were seeded in 96 well plates the day before the experiment. After overnight incubation, cells were treated with V-9302, CB-839, 500 nM rapamycin as a positive control, or the combinations thereof with 10 uM chloroquine for 18 hours. Following treatment, cells were gently washed with assay buffer followed by addition of the dual color detection solution and incubation at 37 ^oC for 30 min. The plate was analyzed on a fluorescence microplane reader (BioTek Synergy 4).