Multiparametric Flow Cytometry for Hematopoietic Stem Cell Characterization

Cryopreserved bone marrow MNCs were thawed and viable cells were counted. One million viable cells were stained with DAPI viability dye, anti-human CD71 (BD Biosciences, clone L01.1), anti-human CD3 (BD Biosciences, clone SP34–2), anti-human CD4 (BD Biosciences, clone L200), anti-CD8b (Thermo, clone 3B5), anti-CD34 (BD Biosciences, clone 563), anti-human CD20 (BioLegend, clone 2H7), anti-CD14 (Miltenyi, clone TUK4), and anti-human CD11b (BD Biosciences, clone ICRF44) for 20 min at room temperature with Human Fc Block (Miltenyi). Alternatively, 200,000 CD34-enriched viable cryopreserved cells were thawed and stained with anti-human CD38 (Caprico Biotechnologies, clone OKT10), CD45RA (BD Biosciences, clone 5H9), CD90 (BD Biosciences, clone 5E10), and DAPI viability dye. Cells were then passed through a 100-micron cell strainer into FACS sorting buffer, run on a FACSAria flow cytometer (BD Biosciences), and analyzed using FlowJo software (BD Biosciences). Compensation controls were run with UltraComp eBeads (Thermo) during each run and fluorescence minus one (FMO) controls were run for each fluorophore in the panel. MPPs were designated as CD34+CD38-CD90-cells and HSCs were designated as CD34+CD38-CD90+ cells, as published previously in macaques.^{110 (link)}