Facs aria cell cytometer

Flow cytometry analysis was performed as described previously^{41 (link)}. For the cell cycle assay, indicated cells were treated with different concentrations of GA for 24 h and harvested for the following steps. A total of 1 × 10⁶ cells per sample were analyzed for cell cycle distribution on a FACS Aria Cell Cytometer (BD Biosciences, San Jose, CA, USA). For the apoptosis assay, 2 × 10⁵ cells per sample were harvested and tested. All data were analyzed using CellQuest software (BD Biosciences).

The flow cytometry analysis was carried out as described previously 41. For cell cycle analysis, HepG2 and BEL7402 cells were treated with vorinostat and/or oxaliplatin for 48 h. A total of 1 × 10⁶ cells per sample were analyzed using FACSAria Cell Cytometer (BD Biosciences, San Jose, CA, USA). For apoptosis analysis, 1 × 10⁵ cells per well were tested. All data were analyzed using CellQuest software (BD Biosciences).

At the endpoint of combination cancer immunotherapy, mice from all groups were killed to harvest splenocytes. After 7 days of splenocytes subculture with 40 ng/mL mouse IL-2 and 20 μg/mL OVA, the splenocytes were cocultured with 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE, Dojindo) - stained live E.G7-OVA cancer cells and NIH3T3 fibroblasts at effector cells/target cells ratio of 10, respectively. In addition, mouse from group f were cocultured CFSE - stained live E.G7-OVA cancer cells and PC-12 cancer cells at effector cells/target cells ratio of 0, 5, 10 and 20, respectively. The cells were then stained with Ghost Dye™ Violet 450 (Bay bioscience) 24 h later. The cytotoxicity of splenocytes against E.G7-OVA cancer cells, NIH3T3 fibroblasts and PC-12 cancer cells were analysed using a FACSAria cell cytometer (BD Biosciences), respectively. The cytotoxicity is calculated by the following formula: cytotoxicity = (total dead target cells−spontaneous dead target cell)/(total target cells−spontaneous dead target cell) × 100%.

At the endpoint of prophylactic and combination cancer immunotherapy, splenocytes were collected from the spleen, milled, vortexed and filtered through a 40-μm cell strainer to obtain single-cell suspension. Anti-CD16/CD32 antibody (2.4G2, Biolegend) with 1/100 dilution was used to prevent the nonspecific staining. Anti-mouse CD8α Ab (BioLegend) and anti-mouse T-Select H-2Kb OVA Tetramer-SIINFEKL Ab (MBL) with 1/50 dilution were used to stain the cells for 30 min. Then, the intracellular cytokine was stained by anti-mouse IFN-γ Ab (BioLegend) with 1/50 dilution. Flow cytometry was performed for the cell suspensions using a FACSAria cell cytometer (BD Biosciences).

For the cell cycle assay, indicated cells were harvested for the following steps. Cells were fixed in 70% ethanol overnight at 4 °C. Then, the fixed cells were resuspended in staining solution (Beyotime, Shanghai) and were incubated for 30 min at 4 °C. A total of 1 × 10⁶ cells per sample were analyzed for cell cycle distribution on a FACS Aria Cell Cytometer (BD Biosciences, San Jose, CA, USA).

The flow cytometry analysis was carried out as described previously 18 (link). For cell cycle analysis, HepG2 and Huh7 cells were treated with MGCD0103 for 48 h. A total of 1×10⁶ cells were analyzed by FACSAria Cell Cytometer (BD Biosciences, CA, USA). For apoptosis analysis, 1×10⁵ cells were analyzed. All data were analyzed by CellQuest software (BD Biosciences, CA, USA).