Flow cytometric analysis was performed as described previously (7 (link), 13 (link), 22 (link)). Briefly, following red cell-lysis (with 0.8% NH4Cl buffer), bone marrow (BM) cells were suspended in FACS buffer (PBS containing 2.5% BSA and 0.5 mM EDTA) and phenotyped using the following antibodies/fluorophores, with gating as described previously (7 (link), 13 (link), 22 (link)) and shown in Supplementary Figure 1 .
LSK HSC analysis involved use of a PE-conjugated lineage cocktail, in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. Antibodies were employed at 0.2 µg/106 cells (1/100 dilution) except for anti-CD45 (1/200 dilution). Streptavidin was used at 1/500 dilution. Cell death was assessed by fixed viability stain (APC-ef780) or 7AAD (BD Bioscience, UK) staining. Data were acquired using a FACS Canto flow cytometer and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) with populations being gated using isotype and fluorescence minus one (FMO) controls (7 (link), 13 (link), 22 (link)).
LSK HSC analysis involved use of a PE-conjugated lineage cocktail, in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. Antibodies were employed at 0.2 µg/106 cells (1/100 dilution) except for anti-CD45 (1/200 dilution). Streptavidin was used at 1/500 dilution. Cell death was assessed by fixed viability stain (APC-ef780) or 7AAD (BD Bioscience, UK) staining. Data were acquired using a FACS Canto flow cytometer and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) with populations being gated using isotype and fluorescence minus one (FMO) controls (7 (link), 13 (link), 22 (link)).
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