Anti mouse cd4 fitc

For cell surface examination, macrophages were incubated in 1% bovine serum albumen (BSA)-PBS buffer at room temperature for 20 min. Thereafter, cells were incubated in the M1 antibody marker CD68 or the M2 antibody marker CD200R (eBioscience, San Diego, CA, USA) in a dark room at 4°C.
For intracellular staining, cells were stimulated with 1 μg/ml brefeldin A (Sigma, USA) for 6 h at 37°C under 5% CO2/95% air atmosphere, and stained for 20 min at room temperature in 0.3% saponin/1% BSA-PBS buffer. After rinsing, cells were incubated for 1 h at 4°C with the following antibodies: iNOS (eBioscience lnc.); arginase 1 (Arg-1) (eBioscience lnc.); Nrf2 (eBioscience lnc.). Expression of iNOS and Arg-1 are key markers associated with M1 and M2 phenotypes, respectively.^{6 (link)}For measurement of T helper 1 (Th1) and regulatory T cells (Treg) cells, anti-mouse CD4 FITC (Abcam) was added and cells were incubated for 30 min at 4°C. Thereafter, cells were incubated in anti-mouse Foxp3 PE (Abcam) and incubated for 30 min at 4°C.
Subsequently, all cells were subjected to flow cytometry detection (Becton Dickinson Biosciences, San Jose, CA, USA) and analysed using CellQuest software.

The peripheral blood samples (300 µL each) were derived from the GEFS+ and the control groups using the lymphocyte separation medium to obtain the peripheral blood lymphocytes, and the lymphocytes were analyzed by flow cytometry. The cells were stained for 30 minutes on ice with the following fluorescence-labeled antibodies (1 µg/2×10⁶ cells) obtained from Abcam: anti-mouse CD3-FITC, anti-mouse CD4-FITC, and anti-mouse CD8-PE. The other two groups (GEFS+ and the control groups) of samples with the same amount of cells were stained for 30 minutes on ice with non-related IgG-FITC as the negative control (background values). The lymphocyte typing was conducted with reference to the product manuals (BBI Life Sciences, Shanghai, China). The cells were washed twice with cold D-Hanks and then more than 10,000 cells from each group were analyzed on BD Accuri C6 [Becton, Dickinson, and Co. (BD), Franklin Lakes, NJ, USA]. Data analysis was performed using BD Accuri C6 software (BD).