Aria facs flow cytometry system

HCT 116 cells were incubated with TCN (0–0.5 μM) for 24h. After that, the cells were collected and washed in PBS and fixed in ice-cold 70% (v/v) ethanol overnight at –20 °C. The cell pellet was resuspended in PBS and stained with a mixture of RNase (10 μg/mL) and PI (50 μg/mL) on sodium citrate containing 0.5% Triton X-100 for 20 min in the dark. Cell cycle distribution analysis was performed using an Aria FACS flow cytometry system (Beckman Coulter, Boulevard Brea, CA, USA).

NCI-H1975 cells were seeded in six-well plates (3 × 10⁵ cells/well), and treated with RPMI-1640 medium with different concentrations (31.3–250 μg/mL) of HMF for 48 h. After HMF treatment, apoptosis was detected using an Annexin V-FITC apoptosis detection kit, according to the manufactures’ protocol. Briefly, cells were treated with different concentrations of HMF, harvested and washed with ice-cold PBS, and then resuspended in 100 μL binding buffer containing 5 μL Annexin V-FITC and 5 μL PI. After incubation for 15 min in the dark at room temperature, cells were analyzed on an Aria FACS flow cytometry system (Beckman Coulter MoFlo XDP, Fullerton, CA, USA).

RAW264.7 cells were treated with LPS (1 μg/mL) or different concentrations (15.6–125 μg/mL) of HMF. After incubation for 24 h, RAW264.7 cells were collected and blocked with PBS containing 5% BSA for 15 min. Flow cytometry buffer (PBS containing 1% BSA) was then added to stop blocking. RAW264.7 cells were stained with PE-conjugated anti-CD40 antibody. Furthermore, RAW264.7 cells were stained with APC-conjugated anti-mouse CD86 antibody or APC-conjugated anti-mouse CD206 antibody to detect M1- or M2-polarized macrophages, respectively, using Aria FACS flow cytometry system (Beckman Coulter MoFlo XDP, Fullerton, CA, USA).