Mouse treg flow kit foxp3 alexa fluor 488 cd4 apc cd25 pe

A total of 25 eight-week-old female BALB/c mice were purchased from the Animal Resources Centre (Nanjing, China). The animals were raised in a constant-temperature environment at 22 ± 2°C, with a regular 12-hour light/dark cycle. Ovalbumin (OVA) and VD3 were obtained from Sigma (St. Louis, Mo, USA). The enzyme-linked immunosorbent assay (ELISA) kits for mouse VD3, IL-4, IL-10, IFN-γ, IL-17, TGF-β, and OVA-IgE were obtained from Abcam (Cambridge, MA, USA). Mouse Treg Flow Kit (FOXP3 Alexa Fluor® 488/CD4 APC/CD25 PE) was purchased from BioLegend, San Diego, CA.

Single cells from the murine spleen were prepared by grinding it, passing it through a 70 µm cell strainer, and suspending it in PBS with 2 mM EDTA and 1% fetal bovine serum (FBS). Red blood cells were lysed by 1× RBC lysis buffer (BioLegend, BD Biosciences, San Jose, CA, USA), and mononuclear cells were washed and cultured in 24-well plates with Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (5000 U/mL), and then stimulated with 50 ng/mL Phorbol 12-myristate 13-acetate (PMA), 500 ng/mL ionomycin (Sigma-Aldrich) and 1× brefeldin A (Thermo Fisher Scientific) for 5 h followed by FACS staining and analysis. Percentages of CD4⁺ IFN-γ⁺ Th1 cells, CD4⁺ IL-4⁺ Th2 cells, CD4⁺ IL-17⁺ Th17 cells in lymphocytes were analyzed by anti-mouse antibodies against FITC/CD4⁺, PE/IFN-γ⁺, APC/IL-4⁺, PE-Cy7/IL-17⁺ (BioLegend). CD25^high FOXP3⁺ Treg cells in lymphocytes were analyzed using Mouse Treg Flow Kit (FOXP3 Alexa Fluor 488/CD4, APC/CD25 PE, BioLegend) by BD FACSVia flow cytometer (BD Biosciences, San Jose, CA, USA).

The excised spleens of the mice were passed through a 70 µm cell strainer to obtain single cell suspension in PBS with 2 mM EDTA & 2% FBS. Red blood cells were lysed, and mononuclear cells were washed and re-suspended in RPMI supplemented with 10% FBS. Percentages of CD25^highFoxp3⁺ Treg cells in lymphocytes were determined using Mouse Treg Flow Kit (FOXP3 Alexa Fluor 488/CD4 APC/CD25 PE, BioLegend) with flow cytometer BD FACSVia (BD Biosciences, San Jose, CA, USA).