B16 Melanoma Cell Apoptosis Assay

B16 cells (7.5 × 10⁵ cells/well) were co-cultured with RAW 264.7 cells (1 × 10⁶ cells/well) by using a 6-well transwell plate [49 (link)]. Briefly, cells were divided into four groups, including blank (only B16 cells), control (B16 cells co-cultured with RAW 264.7 cells), and 25 and 50 μg/mL of TFPS-treated (B16 cells co-cultured with RAW 264.7 cells) groups. After co-culturing for 24 h, B16 cells were collected and detected by Annexin V-FITC/PI apoptosis assay kit (Invitrogen, Carlsbad, CA, USA). Flowsight flow cytometry (Merck, Darmstadt, IN, USA) was used to analyze the apoptosis rate of B16 cells. Caspase-3 activity in B16 cells was performed by the Caspase 3 Activity Assay Kit (Beyotime, Shanghai, China).