Apoptosis Assessment by Flow Cytometry

Assessment of apoptosis was measured by flow cytometry using the active caspase-3 as previously reported (25 (link), 26 (link)). To discriminate viable from non-viable, cells were stained and incubated in FVS V500 dye (BD Bioscience, US) for 15 min at room temperature. For surface staining, 1 × 10⁶ cells were labeled with appropriate antibodies: MHCII (M5/114.15.2), CD11b (M1/70) and F4/80 (PM8) purchased from BD Biosciences (Fanklin Lakes, NJ, US) and eBioscience (San Diego, CA, US). For intracellular staining, cells were washed, fixed and permeabilized with 1X Fixation-Permeabilization buffer and then stained as per manufacturer's instruction with appropriate antibodies: caspase-3 (C92-605) and Bcl-2 (RUO) (all from BD Biosciences, US). After the incubation period cells were resuspended in FACS buffer for acquisition. Acquisition was performed using BD LSRFortessa (BD Biosciences), and data were analyzed using FlowJo software (Treestar, Ashland, OR, US).