Cfse dye

For T cell proliferation analysis, PBMCs from Tg and WT pigs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (BioLegend) as previously described (44 (link)). Briefly, cells were incubated with 5 µM CFSE in PBS containing 5% FBS at 37°C for 5 min and washed three times with ice-cold PBS-5% FBS. 2 × 10⁵ CFSE-labeled PBMCs/well of a 96-well plate were cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin in the presence of stimulation. PBMCs were harvested after a 3-day culture for subsequent analysis by the BD FACSVerse™ flow cytometer.
Peripheral blood mononuclear cells were cultured in 24-well round bottom plates (1 × 10⁶ cells/well) and stimulated for 0, 24, 48, and 72 h with ConA (2 µg/mL), recovered by centrifugation and re-suspended in 100 µL of PBS containing 10% porcine serum. After being washed twice with a cell-staining buffer (BD Biosciences), the cells were suspended in 50 µL of staining buffer and stained with mouse anti-pig 4-1BB monoclonal antibody at 4°C for 30 min. After two washes, they were stained with FITC anti-mouse IgG (BioLegend Cat. No. 406001). Then, the cells were washed two times, suspended in 200 µL of sterile PBS, and analyzed using the BD FACSVerse™ flow cytometer.

First, we collect sorted Teff cells suspension and dilute to CFSE dye (Biolegend, #423801) with PBS to make a 5 mM solution. The Teff cells are labeled in accordance with the instructions provided by the manufacturer. CFSE (Biolegend, #423801)-labeled CD4⁺CD25⁻ Teff cells (4 × 10⁵ cells/mL, 50 μL) alone or cocultured with Treg cells (50 μL) at different ratios (0:1, 1:1,1:2, 1:4, 1:8, Treg: Teff) in the presence of 2 × 10⁶ cells/mL (50 μL) APCs and medium containing 4 μg/mL anti-CD3 antibody (50 μL) in a 96-well U-bottom plate. CFSE dilution was detected three days later, indicating the Treg-cell suppression ability [38 (link), 39 (link)].