Cholesterol quantification

Two methods were used to quantify the cholesterol on T cells. For Filipin III staining, T-Tre/BCN-Lipo-Ava cells were cultured for 12 hours and the CM was collected. Naive and activated cells were incubated with each type of CM for 12 hours. Cells were collected, plated in confocal chambers after centrifugation, and fixed with 4% PFA for 15 min at room temperature. Then, cells were washed twice with PBS and stained with Filipin III (50 μg/ml; Sigma-Aldrich) for 30 min at 4°C. Images were collected using a Zeiss LSM 880 confocal microscope and analyzed using Zeiss ZEN software. In addition, T-Tre/BCN-Lipo-Ava cells, Ava-pretreated T cells, and unconjugated T cells were incubated at 37°C for 12 hours. After incubation, the cells were collected at 0, 2, 4, 8, 12, and 24 hours and stained with Filipin III (50 μg/ml) to quantify the content of plasma membrane cholesterol of T cells over time by flow cytometry.

For oxidation-based cholesterol quantification, activated CD8⁺ T cells were incubated with the CM of T-Tre/BCN-Lipo-Ava cells for 12 hours or with an equivalent dose of free Ava for 12 hours as positive control. The plasma membrane was collected as previously reported (54, 55), and the cholesterol was quantified using an Amplex Red cholesterol assay kit (Invitrogen) according to the manufacturer’s instructions.