Transferrin uptake assay.

CD8⁺ Treg cells were starved in FBS-free medium for 1 hour and incubated with 20 μg/mL FITC-transferrin (Thermo Fisher Scientific, {"type":"entrez-protein","attrs":{"text":"T13342","term_id":"7515367","term_text":"pir||T13342"}}T13342) at 37°C for 10 minutes or indicated time periods. Surface-bound FITC-transferrin was removed with cold acidic buffer (PBS containing 0.5% acetic acid and 500 mM NaCl) for 30 seconds. Cells were washed with cold PBS and fixed with Fix I buffer, followed by detecting the intracellular FITC-transferrin using flow cytometry and/or immunofluorescence staining.