Collected cells were fixed and permeabilized with CytoFix/CytoPerm solution (BD Biosciences) for 1 h at 4 °C. Cells were washed once with Perm/Wash buffer (BD Biosciences) and incubated in the same buffer for 30 minutes at 4 °C. After incubation, cells were centrifuged, stained with rabbit anti-SQSTM1/p62 (1:100; Cell Signaling Technology #5114) or Salmonella O antiserum group B (1:250; BD Biosciences #229481) overnight at 4 °C followed by Alexa Fluor 488-conjugated anti-rabbit (1:500; Cell Signaling Technology #4412) antibodies for 1 h at room temperature. Samples were analyzed using a BD FACScan flow cytometer (BD Biosciences).
For LysoTracker® experiments, YAMC cells were seeded in 24-well plates (1.9 × 105 cells/well) and incubated under non-permissive conditions (37 °C, 5% CO2). The next day, media was replaced with the appropriate drug treatment and cells were returned to the incubator (37 °C, 5% CO2). Twenty-four hours later, cells were stained with 50 nM LysoTracker® Red DND-99 (Invitrogen, Carlsbad, CA) for 30 minutes at 37 °C. Cells were detached from the plates, centrifuged and resuspended in PBS. Samples were analyzed using a BD FACScan flow cytometer (BD Biosciences).
For LysoTracker® experiments, YAMC cells were seeded in 24-well plates (1.9 × 105 cells/well) and incubated under non-permissive conditions (37 °C, 5% CO2). The next day, media was replaced with the appropriate drug treatment and cells were returned to the incubator (37 °C, 5% CO2). Twenty-four hours later, cells were stained with 50 nM LysoTracker® Red DND-99 (Invitrogen, Carlsbad, CA) for 30 minutes at 37 °C. Cells were detached from the plates, centrifuged and resuspended in PBS. Samples were analyzed using a BD FACScan flow cytometer (BD Biosciences).