Fitc rat anti mouse cd34

Mice were sacrificed by cervical dislocation and further decapitated to collect blood. The collected blood was immediately added with EDTA up to the final concentration of 7 mM. Erythrocytes were lysed with 130 mM ammonium chloride followed by the isolation of a mononuclear fraction. A total of 5 × 10⁵ freshly isolated mononuclear cells were incubated with 0.1 μg of Alu–TAMRA DNA for 10 min. Then, 2 μg of FITC rat anti-mouse CD34 (BD, Franklin Lakes, United States), FITC Rat IgG2a κ isotype control (BD, Franklin Lakes, United States) were added; the mixture was incubated for 1 h in the dark at room temperature. Cells were pelleted at 400 g and 4°C for 5 min and washed with PBS (Medigen, Novosibirsk, Russia); flow cytometry was performed on a BD FACSAria™ III Cell Sorter.

BM cells were collected from femurs by flushing with PBS using a syringe, filtering through a 40-μm cell strainer, and treating with ammonium chloride for hemolysis. Bone marrow cells were incubated with FITC Rat anti-Mouse CD34 (#553733, BD Biosciences, San Jose, California, USA) or FITC Rat IgG2a κ Isotype Control (#553929, BD Biosciences) on ice for 60 min. Analyses were then performed using a Cytomics FC500 with FC500 CXP Cytometer software (Beckman Coulter Co., Miami, FL, USA). CD34-positive and CD34–negative bone marrow cells were separated using a MACS Cell Separation instrument (Miltenyi Biotec, Bergisch Gladbach, Germany) with Purified Rat Anti-Mouse CD34 (#553731,BD Biosciences) and Anti-Rat IgG MicroBeads (Miltenyi Biotec).