Flow cytometry was performed with LSRFortessa (Becton–Dickinson). In detail, 2 × 106 cells each were stained in three different multicolor panels, comprised of markers for HSPCs and endothelial progenitor cells (EPCs), immune cell subsets and MSCs and their precursors, followed by RBC lysis (BD Pharm Lyse™ Lysing Buffer, BD Biosciences, Heidelberg, Germany).
For detection and quantification of HSPCs and EPCs, cells were stained with 7-AAD viability dye (BioLegend, San Diego, CA, USA) and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD14-APC-Cy7 (63D3), anti-CD31-Brilliant Violet 605™ (WM59), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-V500 (HI30, BD Horizon™), anti-CD117-Alexa Fluor® 488 (104D2), anti-CD133-Brilliant Violet 421™ (clone 7), and anti-CD309-PE (7D4-6).
For analysis of immune cell subsets, cells were stained with 7-AAD viability dye and the following antibodies (all from BioLegend): anti-CD3-Pacific Blue (HIT3a), anti-CD4-Alexa Fluor® 700 (SK3), anti-CD8-Brilliant Violet 510™ (SK1), anti-CD11b-PE-Cy7 (LM2), anti-CD14-APC-Cy7 (63D3), anti-CD19-PE (HIB19), anti-CD45-FITC (HI30), anti-CD56-Alexa Fluor® 647 (5.1H11), anti-CD183-Brilliant Violet 605™ (G025H7), and anti-CD194-PE-Dazzle™ 594 (L291H4).
MSCs and their precursors were detected in the BM aspirates by staining with 7-AAD viability dye and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD29-APC-Cy7 (TS2/16), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-FITC (HI30), anti-CD73-Brilliant Violet 605™ (AD2), anti-CD90-PE-Cy7 (5E10, BD Horizon™), anti-CD105-PE-CF594 (266, BD Horizon™), anti-CD119-PE (GIR-208), anti-CD146-Brilliant Violet 510™ (P1H12), and anti-CD271-BV421 (C40-1457, BD Horizon™).
Flow cytometry data were analyzed using FCS Express 6 Flow Software (De Novo Software, Pasadena, CA, USA). The percentages of viable cells of each cell type were converted to cell count per milliliter processed BM.
For detection and quantification of HSPCs and EPCs, cells were stained with 7-AAD viability dye (BioLegend, San Diego, CA, USA) and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD14-APC-Cy7 (63D3), anti-CD31-Brilliant Violet 605™ (WM59), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-V500 (HI30, BD Horizon™), anti-CD117-Alexa Fluor® 488 (104D2), anti-CD133-Brilliant Violet 421™ (clone 7), and anti-CD309-PE (7D4-6).
For analysis of immune cell subsets, cells were stained with 7-AAD viability dye and the following antibodies (all from BioLegend): anti-CD3-Pacific Blue (HIT3a), anti-CD4-Alexa Fluor® 700 (SK3), anti-CD8-Brilliant Violet 510™ (SK1), anti-CD11b-PE-Cy7 (LM2), anti-CD14-APC-Cy7 (63D3), anti-CD19-PE (HIB19), anti-CD45-FITC (HI30), anti-CD56-Alexa Fluor® 647 (5.1H11), anti-CD183-Brilliant Violet 605™ (G025H7), and anti-CD194-PE-Dazzle™ 594 (L291H4).
MSCs and their precursors were detected in the BM aspirates by staining with 7-AAD viability dye and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD29-APC-Cy7 (TS2/16), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-FITC (HI30), anti-CD73-Brilliant Violet 605™ (AD2), anti-CD90-PE-Cy7 (5E10, BD Horizon™), anti-CD105-PE-CF594 (266, BD Horizon™), anti-CD119-PE (GIR-208), anti-CD146-Brilliant Violet 510™ (P1H12), and anti-CD271-BV421 (C40-1457, BD Horizon™).
Flow cytometry data were analyzed using FCS Express 6 Flow Software (De Novo Software, Pasadena, CA, USA). The percentages of viable cells of each cell type were converted to cell count per milliliter processed BM.
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