Fitc mouse igg1 isotype control

For EPCs analysis, peripheral blood mononuclear cells were separated using Ficoll (Histopaque 1077; Sigma‐Aldrich, St. Louis, Missouri). Positive cells were double‐stained with anti‐CD34‐FITC (348 053; BD Pharming, San Diego, California) and anti‐KDR‐PE (FAB357P; R&D Systems), anti‐CD34‐FITC and anti‐CD117‐PE (555 714; BD Pharming), and anti‐CD34‐FITC and anti‐CD133‐PE (130‐080‐801; Miltonic Biotic, Bergisch Gladbach, Germany) monoclonal antibodies. Negative controls were also double‐stained with FITC mouse IgG1 isotype control (555 909; BD Pharmingen) and PE mouse IgG1 isotype control (349 043; BD Pharmingen) antibodies. Data analysis was performed using CellQuest Pro software (BD Biosciences). CD34⁺ KDR⁺, CD34⁺ CD117⁺, and CD34⁺ CD133⁺ double‐positive cells were defined as circulating EPCs after gating on the lymphocyte population. The total number of positive cells was calculated based on the absolute leukocyte count × percentage of positive cells (%) and expressed as the absolute number of cells per 1 mL of whole blood.13