Cell Immunostaining and Flow Cytometry Protocol

Cells were stained for immunocytochemistry and flow cytometry as described previously (Perriard et al., 2015 (link)). In brief, for immunocytochemistry, cells were fixed with 4% paraformaldehyde, blocked for 1 hr, then incubated overnight with primary antibodies. The next day, cells were incubated with secondary antibodies before counterstaining with DAPI and slides were mounted with ProLong Diamond Antifade medium (Life Technologies). For flow cytometry staining, cells were first stained with violet Live/Dead (Life Technologies) before staining for extracellular markers with labeled antibodies. For intracellular staining, cells were fixed and permeabilized before adding primary antibodies, then washed and stained with labeled secondary antibodies. See Supplemental Information for detailed methods (Table S2).