Foxp3 fixation permeabilization kit

Intracellular cytokine staining was performed as described earlier [26 (link)]. Briefly, cells were stimulated with 81 nM PMA, 1.34 μM ionomycin, 10.6 μM brefeldin and 2 μM monensin in complete RPMI medium at 37 °C in 5% CO₂ incubator for 6 h. Cells were washed and surface-stained using saturating concentration of specific antibodies on ice for 30 min; washed and incubated with appropriate secondary reagents (1:500 dilution) on ice for 30 min. Intracellular cytokines and transcription factors staining were performed using Foxp3 Fixation/permeabilization kit (Biolegend, San Diego, CA) according to the manufacturer’s instructions.

PBMC suspensions were prepared in FACS buffer (PBS + 1 % FCS + 0.01 % azide) and anti-human CD3, CD4, CD8, CD83, HLA-DR, CD11b, CD33, CD19, CD16 (BD Bioscience, San Jose, CA, USA), CD4, and CD25 (Biolegend, San Diego, CA, USA) were used to determine the relative PBMC immunophenotype. After washing, cells were incubated for 30 minutes at room temperature (RT) with antibody mixtures. Following incubation, cells were washed/fixed with 2 % paraformaldehyde. Intracellular staining with anti-FoxP3 (eBioscience, San Diego, CA, USA) using the FoxP3 fixation/permeabilization kit (Biolegend) was performed according to manufacturer’s instructions. Analysis was performed using the BD LSR-II cytometer, and datasets were analyzed using CellQuest Pro software.

Splenic cells were isolated by gradient centrifugation using lymphocyte separation medium (Dakewe Biotech, Shenzhen, China). Tumor tissues were minced and digested in RPMI containing 2 % FBS, 1 mg/ml type IV collagenase (Sigma Aldrich) and 300 U/ml DNase I (Sigma Aldrich) for 2 h at 37 °C, and passed through a cell strainer to achieve cell suspension.
For surface staining, cells were suspended in staining buffer and incubated for 30 min at 4 °C in the dark with fluorophore-conjugated anti-mouse mAbs: APC-anti-CD3, PerCP-Cy5.5-anti-CD4, PE-anti-CD8, PE-anti-CD19, PE-anti-CD49b, PE-Cy5-anti-CD11b, PE-anti-Gr-1, and APC-anti-PD-L1. For intracellular Foxp3 staining, cells were fixed and permeabilized according to the manufacturer’s protocol and incubated with PE-conjugated anti-mouse Foxp3 antibodies for 30 min at 4 °C in the dark. All antibodies and Foxp3 fixation/permeabilization kit were purchased from Biolegend. Cells were acquired by a flow cytometer (BD LSRII) and analyzed using Flowjo software.