Af488 anti foxp3

Mouse lung lobe tissue weighing 40 mgwas taken from each group at each time point. The tissue was rinsed with PBS, mechanically sheared, ground, and then passed through a 70 μm cell filter. After erythrocytes lysis using ammonium-chloride-potassium lysis buffer (Leagene, Beijing, China, Beijing, China), the cell suspension from a single mouse was used for T-lymphocyte isolation with a mouse organ tissue lymphocyte isolation solution kit (Solarbio, Beijing, China). The isolated cells were washed with PBS, centrifuged, and stained with surface antibodies using PE anti-cluster of differentiation 4(CD4)(Thermo Fisher Scientific, Waltham, MA, USA), APC anti-cluster of differentiation 25(CD25) (Thermo Fisher Scientific), and PE-cy7 anti-IRF4 (Cell Signaling Technology, Danvers, MA, USA) for 45 min. After that, the cells were fixed and permeabilised with a cell-fixing and permeabilisation reagent for 1 h. Subsequently, intranuclear cytokine AF488 anti-FOXP3 (Thermo Fisher Scientific, Waltham, MA, USA) staining was performed. Cells labelled as CD4⁺ CD25⁺ FOXP3⁺ were identified as Tregs, and their expression was assessed using flow cytometry (BD FACS Canto; BD, Franklin Lake, NJ, USA). Flow Jo v10 (BD) software was used for data analysis.