Perfect count microspheres cyt pcm 100

HeLa cells were transfected with specific siRNA. After that, cells were untreated or treated with 10 and 25 nM of MMC for 72 h, medium was discarded, and cells were trypsinized. Cell number and cell cycle was simultaneously analyzed by cell cytometry using Perfect-count microspheres (CYT-PCM-100, Cytognos) and propidium iodide (Invitrogen). Results for survival were expressed in percentage related to untreated controls. The percentage of G2 cells was calculated from the cytometer data using the FlowJo program. When the intensity of the propidium iodide was evaluated in a histogram, the G2 pick was selected and the percentage of cells in G2 was calculated related to the total amount of cells that were evaluated for cell cycle distribution.

Blood, BM cells and splenocytes were collected as previously described^{19 (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 described^{19 (link),40 (link)}. The flow cytometry data were acquired using a FACSCanto II and analysed with FACSDiva (Becton and Dickinson) or FlowJo software.