Anti human cd34 pe cy7

Cells were stained by fluoro-conjugated antibodies for 30 min at 4 °C. After staining, cells were washed with cold PBS for several times, and were resuspended with PBS containing 2% FBS. Cells were analyzed on a FACSCanto II, a FACSAria III or a FACSAria Fusion using the FACSDiva software (BD Biosciences). Analyses were performed using FlowJo (BD). Dead cells were eliminated by DAPI (BioLegend, catalog #422801) or 7-AAD (Miltenyi Biotec, 130-111-568). The following fluoro-conjugated antibodies were used in this study at indicated dilutions: anti-human Lineage Cocktail BV510 (BioLegend, 348807, clone OKT3; M5E2; 3G8; HIB19; 2H7; HCD56, 1:100), anti-human CD34-APCCy7 (BioLegend, 343513, clone 581, 1:500), anti-human CD38-PECy7 (BioLegend, 303515, clone HIT2, 1:500), anti-human CD90-biotin (BioLegend, 328106, clone 5E10, 1:500), anti-human CD45RA-FITC (BioLegend, 304105, clone HI100, 1:100), anti-human CD10-PE (BioLegend, 312204, clone HI10a, 1:100 for sorting and 1:500 for analysis), Streptavidin-BV605 (BioLegend, 405229, 1:500), anti-human CD34-PECy7 (BioLegend, 343616, clone 561, 1:500), anti-human CD19-APC (BioLegend, 302212, clone HIB19, 1:500), anti-human CD19-APC (Miltenyi Biotec, 130-091-248, 1:33), anti-human CD45 VioBlue (Miltenyi Biotec, 130-113-122, clone 5B1, 1:33).

Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies) and then with anti-human CD34 PE-Cy7 (581, BioLegend; 1:100), CD38 Alexa Fluor 647 (AT1, Santa Cruz Biotechnologies; 1;50), CD45RA BV 421 (HI100, BD Biosciences; 1:25), and CD90 BV605 (5E10, BioLegend; 1:30) and analyzed by flow cytometry. For sorting of CD34⁺ or CD34⁺ CD38⁻ CD90⁺ cells, cord-blood-derived CD34⁺ HSPCs were stained directly after isolation from blood with anti-human CD34 FITC (8G12, BD Biosciences; 1:100), CD90 PE (5E10, BD Biosciences; 1:50), CD38 APC (HIT2, BD Bioscience; 1:50), and cells were sorted on a FACS Aria II (BD Bioscience).