2.7. Flow Cytometry

The flow cytometry method has been previously described [9]. For the cell surface marker and Tregs analysis, lymphocytes were isolated from the spleen, and the CD4⁺ T cells were enriched with CD4 (L3T4) MicroBeads mouse isolation kits (Miltenyi Biotec Inc., Auburn, CA 95602, USA). For the analysis of surface markers, the CD4⁺ T cells were stained with APC-conjugated anti-mouse CD4 (GK1.5, 1 : 100), PE-conjugated anti-mouse CD25 (PC61.5, 1 : 100), and FITC-conjugated anti-mouse FOXP3 (3G3, 1 : 100) antibodies from Tonbo Biosciences in PBS containing 2% FBS. The cell membranes were permeabilized, and the nuclear transcription factor FOXP3 was stained using the Transcription Factor Staining Buffer Kit (Tonbo Biosciences, San Diego, CA, USA) according to the manufacturer's instructions. Compensation was performed with a BD LSRFortessa (BD Biosciences). The data were acquired and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).