Foxp3 fixation permeabilization kit

P1 and P21 male and female murine lungs were isolated as described above. Cells were blocked for 30 min with CD16/CD32 (Tonbo Biosciences). For intracellular analyses, cells were stimulated with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (750 ng/mL, Sigma-Aldrich), and GolgiStop (BD Biosciences) for 5 hr, and then surface stained with fluorochrome-conjugated antibodies for 30 min: CD3 (clone: 145–2 C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), and CD8a (clone: 53–6.7, Biolegend). Cells were then permeabilized with FOXP3 Fixation/Permeabilization Kit (BD Biosciences) as indicated by the manufacturer, and stained for TBET (clone: 4B10, Biolegend), GATA3 (clone: L50-823, BD Biosciences), FOXP3 (clone: FJK-16s, eBioscience), RORγt (clone: Q31-378, BD Biosciences), IFNγ (clone: XMG1.2, Biolegend), IL-4 (clone: 11B11, BD Biosciences), IL-10 (clone: JES5-16E3, Biolegend), and IL-17 (clone: TC11-18H10, Miltenyi Biotec) for 30 min. Cells were read using an LSRII flow cytometer using FACSDiva software. Flow data was analyzed using FlowJo (Tree Star Inc). Flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility.

Isolated CD4+ T cell suspensions (1 × 10⁵ cells) were stained with antihuman monoclonal antibodies CD4-FITC and CD25-APC (BD Biosciences, USA), followed by fixation and permeabilization by a FOXP3 Fixation/Permeabilization kit (BD Biosciences, USA) and FOXP3-PE staining (BD Biosciences, USA) according to the manufacturer's instructions. Cells were then analyzed using a flow cytometer (FACS Canto II; BD Biosciences, San Jose, CA, USA). The percentage of CD4-, CD25-, and FOXP3-positive cells was calculated and analyzed using a FlowJo7.6.5 software (TreeStar Inc., USA).

Lymphocytes were isolated from the C57BL/6 mouse spleens and MLNs in single-cell suspensions. The cells were stained with fluorescence-labeled antibodies against CD4-FITC and CD44-APC at 4°C for 30 min. The samples were stained with antibodies against Foxp3-PE (NRRF30, eBioscience Inc., San Diego, CA, USA) at room temperature for 30 min in the dark after using a Foxp3 fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA) [29 (link)]. The lymphocytes collected from the Foxp3^GFP mouse spleens were stained with fluorescence-labeled antibodies against CD4-PE and CD62L-APC at 4°C for 30 min. Flow cytometry was performed on an LSRII cytometer (BD Biosciences) and analyzed with the FlowJo 7.6 software. Anti-mouse CD4-FITC (GK1.5) and Foxp3-PE (NRRF30) were purchased from eBioscience. CD62L-APC (MEL-14) was purchased from BD Biosciences. CD4-PE (GK1.5) and CD44-APC (IM7) were obtained from BioLegend Inc. (San Diego, CA, USA).