Flowsight flow cytometry

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).

Phagocytic uptake of RAW 264.7 cells was detected by using FITC-dextran. RAW 264.7 cells (1 × 10⁶ cells per well) were seeded in a 6-well plate and incubated for 24 h. Cells were then treated with S2, S4 and S5 for 2 h, followed by co-treatment with LPS (1 μg/mL) for 24 h. After discarding the supernatant, cells were incubated with fresh medium containing FITC-dextran (1 mg/mL) at 37 °C for 1 h. The phagocytosis of cells on FITC-dextran were analyzed by Flowsight flow cytometry (Merck, Schwalbach, Germany).

The phagocytic ability of RAW 264.7 cells was measured by FITC-dextran [51 (link)]. RAW 264.7 cells (1 × 10⁶ cells per well) were seeded in a 6-well plate and incubated for 24 h. Then, cells were treated with different doses of TFPS (1, 12.5, 25 μg/mL) or 1 μg/mL of LPS for 24 h. After discarding the supernatant, the cells were incubated with a fresh medium containing FITC-dextran (1 mg/mL) at 37 °C for 1 h. The phagocytosis of FITC-dextran in RAW 264.7 cells was analyzed by Flowsight flow cytometry (Merck, Darmstadt, IN, USA).