Multiparameter Flow Cytometry Analysis

Flow cytometry analyses were performed using a BD Fortessa LSRII flow cytometer (BD Biosciences). Cells were prepared from peripheral blood, bone marrow (through flushing by centrifugation), and spleen (through crushing), lysed in red blood cell lysis buffer (155 mM NH₄Cl, 12 mM NaHCO₃, 0.1 mM EDTA) for 5 minutes at 4°C and washed in PBS + 1% FBS before staining. Bone marrow and spleen cells were lineage depleted using the stem cell technologies hematopoietic progenitor isolation kit as per the manufacturer’s instructions (StemCell technologies #19856) before staining. Cells were stained with cell surface antibodies (key resources table) in PBS + 1% FBS for 1 hour at 4°C, washed 3 times in PBS + 1% FBS and resuspended in PBS + 1% FBS + 4′,6-diamidino-2-phenylindole (0.1 μg/mL) before analysis by flow cytometry. For intracellular flow cytometry, cells were first stained with cell surface antibodies and fixable live dead dye (either Zombie UV Biolegend or Sytox Green Life Technologies) followed by fixation in 1.6% formaldehyde (Sigma) at room temperature for 10 minutes in the dark and permeabilized in 1 mL Perm buffer III (BD biosciences) on ice for 30 minutes in the dark. Cells were washed 3 times in PBS + 1% FBS and incubated with intracellular antibodies for 4 hours at 4°C. Stained cells were washed three times in PBS + 1% FBS and analyzed by flow cytometry.