Anti flk1 pe cy7

Spleen cells were stained with anti-CD45-FITC (BD Bioscience, Franklin Lakes, USA), anti-sca1-BV421 (BD Bioscience, Franklin Lakes, USA), anti-flk1-PE-Cy7 (BD Bioscience, Franklin Lakes, USA), anti-B220-APC (BD Bioscience, Franklin Lakes, USA), anti-IgM-Per-CP/Cy5.5 (BD Bioscience, Franklin Lakes, USA), anti-CD11b-APC/Cy7 (BD Bioscience, Franklin Lakes, USA) and anti-CD5-PE (BD Bioscience, Franklin Lakes, USA). Dead cells were identified by using a Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego USA) and doublets were excluded.
The following fractions were sorted by using flow cytometry (FACSAria, BD): CD45⁺, sca1⁺/flk1⁺ (sca1⁺/flk1⁺ cells); CD45⁺, B220⁺/IgM⁺, CD11b⁻/CD5⁻ (B2 cells); CD45⁺/sca1⁻/flk1⁻, B220⁺/IgM⁺, CD11b⁻/CD5⁻ (sca1⁻/flk1⁻ B2 cells).

Flow cytometry analysis was performed as previously described [28 (link), 30 (link)]. Samples were measured on a FACSCalibur or FACSCanto II (BD Bioscience, Franklin Lakes, USA) and analyzed with FlowJo (Tree Star, Ashland, USA). Dead cells were identified by using a Zombie Aqua™ Fixable Viability Kit (Biolegend, San Diego USA). Doublets were excluded in every analysis. Samples were stained with anti-sca1-APC (R&D Systems, Minneapolis, USA) anti-flk-1-PE (BD Bioscience, Franklin Lakes, USA), anti-B220-APC (BD Bioscience, Franklin Lakes, USA), anti-IgM-Per-CP/Cy5.5 (BD Bioscience, Franklin Lakes, USA), anti-CD11b-APC/Cy7 (BD Bioscience, Franklin Lakes, USA), anti-CD5-PE (BD Bioscience, Franklin Lakes, USA), anti-sca1-FITC (BD Bioscience, Franklin Lakes, USA), anti-flk1-PE-Cy7 (BD Bioscience, Franklin Lakes, USA), anti-CD3-FITC (BD Bioscience, Franklin Lakes, USA), anti-CD4-PE/Cy7 (eBioscience, Waltham, USA), anti-CD8-APC (Biolegend, San Diego, USA), anti-sca1-PerCP/Cy5.5 (eBioscience, Waltham, USA), anti-CD11b-PE (BD Bioscience, Franklin Lakes, USA), anti-CD115-APC (Biolegend, San Diego USA), anti-Gr1-FITC (BD Bioscience, Franklin Lakes, USA) and anti-CD45 FITC (BD Bioscience, Franklin Lakes, USA).