Percp cy 5.5 hamster anti mouse cd3e

The positive rates of CD4⁺ and CD8⁺ T lymphocyte subsets were analyzed by flow cytometry. Briefly, 3 ml of red blood cell lysis buffer was added to each sample to completely lyse red blood cells. Then, the samples were washed and re-suspended with DMEM to 1 × 10⁶ cells·ml^-1. The cells were transferred to 48-well plates with 100 μl volume of each well, and 2 μl of cell activation cocktail (BD Biosciences) and 1 μl of BrefeldinA (BD Biosciences) were added to each sample and incubated at 37°C for 6 h. After washing and re-suspension with phosphate-buffered saline, the samples were incubated with specific fluorescent antibodies (BD Biosciences) of PerCP-Cy™5.5 Hamster Anti-Mouse CD3e (0.5 μl/sample), FITC Rat Anti-Mouse CD4 (0.2 μl/sample), and APC-H7 Rat anti-Mouse CD8a (0.5 μl/sample) for 30 min at room temperature in the dark according to the manufacturer’s guidelines. All samples were stained in triplicate. The samples were analyzed by using the CytoFLEX flow cytometer (Beckman Coulter Life Sciences), and the data were analyzed by the CytExpert software (Beckman Coulter Life Sciences).

The positive rates of CD4⁺, CD8⁺ T lymphocyte subsets were analyzed by flow cytometry. Briefly, 3 mL of red blood cell lysis buffer was added to each sample to completely lyse red blood cells. Then the samples were washed and re-suspended with DMEM to 1 × 10⁶ cells/mL. Cells were transferred to 48-well plates with a 100 μL volume of each well, 2 μL of cell activation cocktail (BD biosciences) and 1 μL of BrefeldinA (BD biosciences) were added to each sample and incubated at 37°C for 6 h. After being washed and re-suspended with PBS, samples were incubated with specific fluorescent antibodies (BD biosciences) of PerCP-Cy™5.5 Hamster Anti-Mouse CD3e (0.5 μl/sample), FITC Rat Anti-Mouse CD4 (0.2 μl/sample), and APC-H7 Rat anti-Mouse CD8a (0.5 μl/sample) for 30 min at room temperature in the dark according to the manufacturer’s guidelines. All samples were stained in triplicate. The samples were analyzed by the CytoFLEX flow cytometer (Beckman Coulter Life Sciences), and the data were analyzed by the CytExpert software (Beckman Coulter Life Sciences).

The anticoagulated blood sample (100 μl) from each group was blocked with 0.4 μg anti-mouse CD16/32 antibody (Biolegend) for 15 min at room temperature (RT), followed by incubation with 0.2 μg fluorescent-labeled detection antibodies for 2 h at 4°C in the dark. T cells were stained with the following antibodies: PerCP-Cy5.5 Hamster anti-mouse CD3e (BD Biosciences), FITC Rat anti-mouse CD4 (BD Biosciences), PE Rat anti-mouse CD8a (BD Biosciences) and Isotype control antibodies (BD Biosciences). Erythrocytes in blood samples were lysed with 500 μl Red Blood Cell Lysis Buffer (Tiangen) on ice in the dark. The treated samples were then centrifuged at 500 × g for 10 min. Then, the pellet was re-suspended in 400 μl PBS. The suspensions were analyzed using a BD Accuri^TM C6 Plus flow cytometer (BD Biosciences). All data sets were analyzed with Flowjo software (TreeStar, Ashland, OR, United States).