In a 1.5 mL tube, cells were washed with ice cold phosphate-buffered saline (PBS). For (CD) cell surface markers, cells were stained with PE-CD24 (#555428, BD Biosciences), or APC-CD44 (#559942, BD Biosciences) or APC-CD52 (Clone HI186, BioLegend) in PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) on ice in the dark for 30 min. For subsequent intracellular staining, cells were fixed in 1% paraformaldehyde (PFA) for 10 min followed by permeabilization using 0.5% TritonX100 in PBS for 10 min at room temperature. Cells were stained with primary antibodies rabbit anti-GATA1 (1:400, Cell Signaling, D52H6), mouse anti-GATA2 (1:100, Abnova, H00002624-M01), rabbit anti phospho c-JUN II (Ser63, Cell Signaling), or mouse or rabbit IgG as isotype control in PBS containing 0.5% TritonX100, 2 mM EDTA and 0.5% BSA (Sigma) for 1 h at room temperature. After washing with staining buffer, cells were labeled with Alexa-conjugated donkey anti-mouse or anti-rabbit Alexa 488 or Alexa 647 antibodies (life technologies) at a dilution of 1:500 for 30 min at room temperature. Finally, cells were washed and sorted for CD24 or analyzed using the BD FACSAriaII.
Flow cytometric analysis and statistics were performed using FlowJo V.10.0.8.
Flow cytometric analysis and statistics were performed using FlowJo V.10.0.8.