Fitc conjugated anti cd34 antibody

The numbers of EPCs in each sample were assessed according to a previously published technique (12 (link), 13 (link)). Circulating EPCs were measured using flow cytometry and quantified as PBMNs co-expressing CD34 (a hematopoietic stem and EPC marker) and CD309 (a vascular endothelial growth factor receptor). In brief, we incubated 200 μl of PBMNs with 5 μl of FITC-conjugated anti-CD34 antibody (eBioscience, San Diego, USA) and PE-conjugated anti-CD309 antibody (eBioscience, San Diego, USA) in the dark for 15 min. After incubation, each sample was washed twice with FACS buffer. Approximately 10⁴ events per sample were run on a Guava^Ⓡ easyCyte 8 Flow Cytometer (Millipore Corporation, Billerica, USA), and samples were analyzed with FlowJo software (Tree Star, Ashland, USA).

Enzymatically dissociated
cells were rinsed once with KG2⁺ and stained with fluorescein
isothiocyanate (FITC)-conjugated anti-CD34 antibody (eBioscience)
and phycoerythrin (PE)-conjugated anti-α6 integrin antibody
(eBioscience) for 30 min on ice. After two washes with FACS buffer
(PBS containing 2% BSA, 2 mM EDTA), cells were analyzed in a flow
cytometer (LSRFortessa X-20). The CD34-positive cells were quantified
using the FlowJo software version 10. The marker expression was examined
using live cells after the exclusion of cell doublets and dead cells
stained with 7AAD (eBioscience).