Anti mouse cd3 pe cyanine7

To quantify the percentage of hematopoietic stem cells (HSCs), BM cells (1 × 10⁵ cells) were stained with anti-mouse CD117 (c-Kit) FITC and anti-mouse Ly-6A/E (Sca-1) PE antibodies (eBioscience, San Diego, CA, USA). The percentages of CD3⁺CD4⁺ (helper T cell, Th) and CD3⁺CD8⁺ (cytotoxic T cell, CTL) lymphocytes were also measured. After 12 h or 24 h of treatment, BM cells were harvested and stained with anti-mouse CD3 PE-Cyanine7, CD4 PE-Cyanine5 and CD8a PE antibodies (eBioscience, San Diego, CA, USA). The percentages of staining cells were counted and analyzed by FACS Calibur cytometer and CellQuest software (Beckman Coulter, Brea, CA, USA). Each experiment was performed in triplicate with three replicates each.

The percentages of apoptotic cells in BMCs were measured using an Annexin V-FITC Apoptosis Detection kit (eBioscience Inc, San Diego, CA, USA). Briefly, cells were washed twice with precooled PBS, and then were resuspended in 500 μL of binding buffer at a concentration of 1×10⁶ cells/mL. After adding 5 μL of Annexin Ⅴ-FITC solution and propidium iodide (PI) (1 μg/mL), the cells were incubated for 15 min at room temperature. The apoptosis rates of BMCs were analyzed by flow cytometry. To quantify the percentages of CD3⁺CD4⁺ cells, BMCs were washed and stained with anti-mouse CD3 PE-Cyanine7 (0.5 μg, 1×10⁵ cells/test) and CD4 PE-Cyanine5 (0.06 μg, 1×10⁵ cells/test) antibodies (eBioscience, San Diego, CA, USA). Anti-mouse CD4 PE-Cyanine5, IFN-γ PE (0.25 μg, 1×10⁵ cells/test), IL-4 PE-Cyanine7 (1 μg, 1×10⁵ cells/test) and anti-mouse/rat IL-17A FITC antibodies (0.25 μg, 1×10⁵ cells/test) (BD Bioscience) were used to determine the percentages of cells that were Th1, Th2 and Th17 cells. Anti-mouse CD4 PE-Cyanine5, anti-mouse CD25 PE (0.125μg, 1×10⁵ cells/test) and anti-mouse/rat-FOXP3-FITC antibodies (1 μg, 1×10⁵ cells/test) (eBioscience) were used to determine the percentages of Treg cells. Flow cytometry was performed using a Facs Calibur cytometer. CellQuest software (Beckman Coulter, Brea, CA, USA) was used to analyze the data.