Intracellular Lipid ROS Measurement

Flow cytometry analysis was performed for lipid ROS measurement (Yu et al. 2023 (link)). HCC cells were grown in 6-well plates at 2 × 10⁵ cells/well. After treatment with 2 µM erastin or an equal volume of DMSO for 12 h, the culture medium received clearing and washing with PBS. Next, HCC cells received staining with BODIPY-C11 (5 µM) for 20 min at 37 °C. After washing twice with PBS and filtering with a 0.4-μM cell filter, flow cytometry was used to assess intracellular lipid ROS. The iTreg cells were generated in vitro. The transfected HCC cells received coculture with CD4⁺ T cells for 48 h, and proliferation of CD4⁺ T cells received assessment by CFSE staining using flow cytometry. Antibodies including APC-anti-CD4 or PE-Cy7-anti-CD4 APC-anti-CD25, PE-anti-FOXP3, PE-anti-CTLA4, PE-anti-TIGIT, PE-anti-TNFRSF4, or isotype controls provided by Invitrogen (USA) were utilized for analyzing the phenotype of various Tregs. FOXP3/CD25/CTLA4/TIGIT/TNFRSF4 staining was performed with Fix/Perm buffer (Invitrogen, USA). After staining, cells received washing and fixation with 2% paraformaldehyde before analysis through flow cytometry. The results were analyzed using FlowJo software. There are three biological replicates for flow cytometry analysis.