Flow cytometric analysis was performed as described previously (11 (link), 14 (link), 30 (link), 33 (link)). Briefly, following red cell-lysis (with 0.8% NH4Cl buffer), BM, blood, spleen, MLN and adipose tissue cells were suspended in FACS buffer (PBS containing 2.5% BSA and 0.5 mM EDTA) and phenotyped using the following antibodies:
HSC analysis involved use of a dump channel (PE-conjugated lineage cocktail) in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. For surface marker staining, antibodies were used 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 FACS Canto or BD LSRII flow cytometers and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) and populations were gated using isotype and fluorescence minus one (FMO) controls (11 (link), 14 (link), 30 (link), 33 (link)). Exemplar gating strategies are shown inSupplementary Figure 1
HSC analysis involved use of a dump channel (PE-conjugated lineage cocktail) in combination with FITC anti-Sca-1 and APC or biotin anti-CD117 antibodies. For surface marker staining, antibodies were used 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 FACS Canto or BD LSRII flow cytometers and analysed using FlowJo Software (Tree Star In, OR USA, version 8.8.7) and populations were gated using isotype and fluorescence minus one (FMO) controls (11 (link), 14 (link), 30 (link), 33 (link)). Exemplar gating strategies are shown in
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