LC3 punctate and LAMP2 staining and quantification were performed as described previously12 (link). After treatment, cells on coverslips were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and immunostained with primary Abs (anti-LC3; PM036, MBL International, 1:400) or (anti-LAMP2; sc-5571, Santa Cruz, 1:400) for 2 h. Cells were washed to remove primary Abs and incubated with the fluorescently labeled secondary Ab (Alexa 488-conjugated anti-rabbit IgG; A17041, Molecular Probes, 1:400) for 2 h. Nuclei were stained by incubation with DAPI (D9564, Sigma) for 5 min. After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (LSM 710, Zeiss, Thomwood, NY, USA).
LC3 punctate dot fluorescence intensity was measured using ImageJ analysis software. For colocalization analysis, the co-distribution of LC3 autophagosomes or LAMP2 phagolysosomes with bacterial phagosomes were quantified using the ImageJ analysis software. Each condition was assayed in triplicate, and at least 100 cells per well were counted.
LC3 punctate dot fluorescence intensity was measured using ImageJ analysis software. For colocalization analysis, the co-distribution of LC3 autophagosomes or LAMP2 phagolysosomes with bacterial phagosomes were quantified using the ImageJ analysis software. Each condition was assayed in triplicate, and at least 100 cells per well were counted.