Quantifying Stem Cell Markers in PBMCs

Single-cell suspensions (1 × 10⁶) of PBMC were analyzed for expression of lineage-negative (Lin−) CD34+ and CD133+ stem cell markers as we described^{24 (link),25 (link)}. All antibodies used were from commercial sources: CD133 (Abcam, Cambridge, U.K.); CD34 (R&D Systems, Minneapolis, MN); Cy3 (rabbit), FITC (goat) (eBioscience). Nonspecific antibody binding was blocked with donkey and mouse serum (Sigma-Aldrich) for 30 min. Cells were incubated with antibodies for 1 h at 4 °C, and the positive cells were counted by flow cytometry (fluorescence-activated cell sorting [FACS]) using CELLQuest software (Becton Dickinson, Franklin Lakes, NJ).