Foxp3 perm buffer

This test was carried out using the previously described method^{25 (link)}. Briefly, all treated/untreated cell lines were cultured on sterile cover slip sets at the bottom of each 6-well cell culture plate (cell density 120 × 10⁴/each well). At the end of each time point (12, 24, and 48 h), the cells were fixed by 4% paraformaldehyde (PFA) for 5 min and were permeabilized by 0.1% Triton X-100 for 5 min. Then, 50 µl of diluted 4′,6-diamidino-2-phenyl indole (DAPI) was diluted 1:2000 with its buffer (FOXP3 Perm Buffer, BioLegend, San Diego, USA) and added to each well. After 5 min incubation at room temperature, the cells were washed with sterile PBS (pH 7.2). The stained cells on cover slips were reversely placed on the slides and then were analyzed using a fluorescent microscope (Olympus BX61, Center Valley, PA, USA) equipped with a U-MWU2 fluorescence filter (excitation filter BP 330–385, dichromatic mirror DM 400, and emission filter LP 420).

Prediluted antibodies were prepared for staining of cell surface markers. For intracellular staining, cells were permeabilized in Foxp3 Fix/Perm solution (BioLegend) and incubated with anti-Foxp3 antibodies diluted in Foxp3 Perm buffer (BioLegend). The antibodies used: anti-CD3-PerCP (clone 145-2C11, BioLegend), anti-CD4-Pacific blue (clone RM4-5, BioLegend), anti-Foxp3-Alexa Fluor 488 (clone FJK-16s, Thermo Fisher Scientific), and anti-Nrp-1-APC (clone 3DS304M, Thermo Fisher Scientific). FACSCanto II (BD Biosciences) and FlowJo software (BD Biosciences) were used to collect and analyze the data.

All collected peritoneal fluid samples were centrifuged at 1600 rpm, at 4 °C for 9 min, then supernatant was discarded, and cells were fixed with 4% par formaldehyde (Sheng Gong, Shanghai, China) for 35 min, at 4 °C in the dark. Then cells were washed twice with phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). After centrifugation and removal of the supernatant, the fixed cells were resuspended in Foxp3 Perm Buffer (10 × , Biolegend, San Diego, USA) according to the manufacturer protocol. Finally, these cells were labeled with flow cytometry antibodies according to the manufacturer protocol and detected by flow cytometry.

To assess proliferation, CD4⁺ T cells were stained with either 1 µM of CTV or CTFR (both from Life Technologies), before stimulation and culture. To assess intracellular cytokine expression after cell culture, cells were stimulated for 3 h in the presence of PMA (50 ng/mL, Sigma‐Aldrich), ionomycin (750 ng/mL, Sigma‐Aldrich), and GolgiStop (BD Biosciences); GolgiPlug (BD Biosciences) was additionally added for complete blocking of TNF export when measuring expression of mTNF. Cells were labelled with a fixable viability dye (LIVE/DEAD fixable dead cell stains, ThermoFisher Scientific). Cells were subsequently washed and stained extracellularly, followed by fixation with 2% PFA (paraformaldehyde, Sigma‐Aldrich), permeabilization using the FOXP3 perm buffer (BioLegend). Cells were stained with various combinations of the following fluorescently conjugated antibodies (see Supporting Information Table 1). Stained cells were acquired using a FACSCantoII or LSRFortessa (BD Biosciences); in most experiments, 100 000 T cell events were recorded. All flow cytometry data were analysed using FlowJo software (version 10, Tree Star, Ashland, USA). Representative CD4⁺ T cell gating strategy is shown in Supporting Information Fig. 2. Guidelines for the use of flow cytometry in immunological studies were adhered to 38.

NK cells were stained for 20 min (37 °C/5% CO₂) with 7-AAD (A1310, Thermo Fisher Scientific) to distinguish live and dead cells. Cells were fixed using 1% paraformaldehyde, washed with FACS buffer (0.09% sodium azide and 2% FBS in DPBS), and permeabilizated with FOXP3 Perm buffer (353097, BioLegend) for 20 min at room temperature. Cells were then resuspended, incubated for 30 min at room temperature in the dark with a fluorochrome-conjugated antibody, and washed twice with FACS buffer. Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) and data were analyzed using CytExpert (Beckman Coulter) and FlowJo software (Treestar Inc, Ashland, Oregon). The antibodies used are listed in Additional file 1: Table S3.