Blood, BM cells and splenocytes were collected as previously described19 (link),40 (link). The different cell samples (0.5–2 × 106 cells) were stained for the indicated surface markers (see Supplemental Table S2 online) for 20 min at room temperature and subsequently washed twice with buffer A. We distinguished live and dead cells by adding SYTOX Green (S7020, Life Technologies) or DAPI (D9542, Sigma-Aldrich) 5 min before FACS analysis. Haematopoietic progenitors were isolated from the spleen and BM by magnetic-activated cell sorting (MACS) using a Lineage Cell Depletion Kit (130-090-858, Miltenyi) and MACS separation MS columns (130-042-201, Miltenyi) prior staining. Unstained cells were used as a negative control to establish the flow cytometer voltage settings, and single-colour positive controls were used to adjust compensation. The absolute number of cells was calculated by adding Perfect-Count Microspheres (CYT-PCM-100, Cytognos) to the flow cytometry samples. Apoptosis, cell cycle, and cell proliferation were determined by flow cytometry as previously described19 (link),40 (link). The flow cytometry data were acquired using a FACSCanto II and analysed with FACSDiva (Becton and Dickinson) or FlowJo software.
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