Cd34 alexa647

Dissociated single cells in PBS/BSA/% EDTA were incubated with various antibody combinations for multiparameter flow acquisition and analysis, as previously described (Patel et al., 2017 (link)). The following combinations of antibodies were used to assess endothelial progenitor cell populations: Rat anti-mouse VE-Cadherin FITC, VEGFR2 PE, CD31 PE-Cy7, CD34 Alexa647, and CD45 V450 (Becton Dickinson).

Dissociated single cells in FACS buffer (0.5% BSA, 1 mM EDTA in 1x PBS) were then incubated with various antibody combinations for FACS analysis and sorting. A Gallios™ flow cytometer was used for sample acquisition, with subsequent data analyses performed with Kaluza^® analysis software (Beckman-Coulter, Miami, Florida, USA). A FACSaria cell sorter was utilised for FACS sorting using the FAVSDiva v5.0.3 software with subsequent analysis performed with FlowJo v10 (Becton Dickinson, Franklin Lakes, NJ, USA). Standard doublets discrimination and 7′AAD or FVS live/dead staining was carried out to only include live singlet events. The following combinations of antibodies were used to assess the endothelial hierarchy populations: Rat anti-mouse VEGFR2 PE (1:100), CD31 PE-Cy7 (1:1000) and CD34 Alexa647 (1:100) (Becton Dickinson, NJ, USA), Rat anti-mouse Lineage cocktail BV450 (1:50) (Biolgend), CD26 PE (1:200), CD140α BV605 (1:100) and Rat anti-mouse CD144 FITC (1:50) (eBioscience).

Dissociated single cells in PBS/BSA/EDTA were then incubated with various antibody combinations for multi-parameter flow acquisition and analysis. A Gallios™ flow cytometer was used for sample acquisition, while unbiased data analyses were performed with Kaluza® analysis software (Beckman-Coulter, Miami, FL, USA). FACS was performed by using a FACSaria cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA). Extreme care was taken during cell sorting to ensure only “singlets” were being gated and any potential “doublets” were being gated out. Cell populations were collected in 5 ml polypropylene tubes containing 100% fetal calf serum (FCS). The following combinations of antibodies were used to assess the endothelial hierarchy populations: Rat anti-mouse VEGFR2 PE (1:50), Sca-1 (1:50), c-KIT (1:50), CD31 PE-Cy7 (1:100), and CD34 Alexa647 (1:50) (Becton Dickinson, NJ, USA), Rat anti-mouse Lineage cocktail BV450 (1:50) (BioLegend), Rat anti-mouse CD144 FITC (1:50) (eBioscience). FMO was used to delineate negative gating for each antibody.