Guavasoft analysis software

Cells were seeded at a concentration of 1 × 10⁶ cells per well of 10 mm plate. Following incubation, samples were prepared as previously described^{56 (link)} and analyzed for DNA content using the Guava EasyCyte™ flow cytometer and the GuavaSoft analysis software (Millipore, Watford,UK).

The CAL27 cells were added to flow cytometry tubes along with 2 mL of fluorescence-activated cell sorting (FACS) buffer and centrifuged at 400 g for 10 min to wash the cells. The cells were then treated with 100 µL Fc receptor blocking buffer for 15 min at 4 °C. Thereafter, the cells were incubated with 2 mL of FACS buffer and mouse anti-human PD-L1 phycoerythrin (PE)-conjugated monoclonal antibody (R&D systems, USA) or mouse lgG2A PE-conjugated monoclonal antibody (control) (R&D systems; USA) in the dark for 30 min. The molecular expression levels of PD-L1 on the surface of the cells were measured using a flow cytometer (Guava Flow Cell for easyCyte systems; Millipore, Burlington, MA, USA), and the GuavaSoft analysis software (Millipore) was used for data analysis. Apoptotic levels of the CAL27 cells were evaluated using an apoptosis test kit (Xinbosheng, China) according to the manufacturer’s instructions.

Cells were seeded at a concentration of 1 × 10⁵ cells per well of a 60-mm plate and treated with TPGS or YM155 as indicated. Cells were harvested and stained as described by Alexa Fluor^TM 488 Annexin V/Dead Cell Apoptosis kit (Life Technologies). Cell viability, death and apoptosis were evaluated using the Guava EasyCyte™ flow cytometer and the GuavaSoft analysis software (Millipore, Watford,UK). The annexin-V positive/PI negative cells were recognized as early apoptotic cells by the cytometer software whereas the annexin V positive/PI positive cells were identified as late apoptotic/dead cells. Similarly, the annexin V-negative/PI negative cells were identified as viable cells.