Isolation and Characterization of Pre-HSCs

Cell suspensions were stained with following antibodies: Ter119-v500 clone (Clone TER-119); anti-CD41-BV421 (brilliant violet 421) or Alexa Fluor 488 (clone MW30reg); anti-CD45 FITC (clone 30-F11); biotinylated anti-VE-cadherin (clone 11.D4.1) followed by incubation with streptavidin-APC (all purchased from BD Pharmingen or BioLegend). Anti-mouse VE-cadherin (VC) antibody was biotinylated in-house using the FluoReporter Mini-Biotin-XX Protein Labeling Kit (Invitrogen). Cell populations were sorted using a FACSAria II sorter (BD) followed by purity checks. Type I pre-HSCs (Ter119⁻VC⁺CD41⁺CD45^-) and type II pre-HSCs (Ter119⁻VC⁺CD41⁺CD45⁺) were sorted from E11 AGMs as described previously (Rybtsov et al., 2011 (link)). Dead cells were excluded using 7AAD staining and fluorescence minus one control staining was used to define gating strategies. Data acquisition and analyses were performed using Fortessa (BD) and FlowJo software.