Fatty acid uptake

Fatty acid uptake was assessed by the uptake of fluorescently labeled fatty acids using the QBT Fatty Acid Uptake Assay Kit (R8132, Molecular Devices, CA, USA), according to the manufacturer's instructions. After the experimental treatment, all culture medium was removed from each well and replaced with culture medium in the absence (negative control) or presence of fluorescent fatty acids (final concentration of 300 μg/ml) and incubated at 37°C with 5% CO₂ for 10 min before flow cytometry analysis. The uptake reaction was stopped by removing the incubation medium and washing the cells twice with pre-cold 1 × PBS (Solarbio, Beijing, CN). Cells were collected by centrifugation at 3,000 rpm for 5 min and subsequently resuspended in 400 μl pre-cold 1 × PBS and maintained at 4°C for later flow cytometry analysis performed within 30 min. For each measurement, data from 10,000 single-cell events were collected using a BD LSRFortessa flow cytometer (BD Biosciences, CA, USA), and each flow cytometric measurement was digitized as fluorescence intensity which was corrected by the negative control.