Autophagy analysis

Autophagy induction in THP1 LC3GFP expressing cells was analyzed as described previously [67]. Briefly, cells were permeabilized with 0.05% saponin for 5 minutes, washed and resuspended in PBS containing 5% FCS. Permeabilization resulted in loss of cytosolic LC3I while LC3II bound to autophagosome membranes were retained, which was measured by flow cytometry (50,000 cells acquired, BD Accuri C6). For immunofluorescence analysis, bacteria were stained with 0.4mg/ml AF647-NHS ester (Molecular Probes) in 0.1M sodium bicarbonate solution for 30 minutes at 37°C and used for infecting cells on slides. At specified time points, cells were fixed with 4% paraformaldehyde overnight, stained with Hoechst 33342 and analyzed by confocal microscopy (Zeiss LSM710).