Evaluating THLE-2 cell immune markers

The detailed procedures were previously described [56 (link)]. THLE-2 cells (2 × 10⁵) were treated with 100 µg/mL IPIL or 3:1 ratio of THLE-2 cell culture medium:T-CM. Twenty-four hours after the treatment, THLE-2 cells were trypsinized, pelleted, and washed 2× with 1× FACS buffer (1 × PBS + 1% FBS). The THLE-2 cells were then stained with the following antibodies including IgG isotype controls: PD-L1 FITC (BD Bioscience, cat# BD558065, San Jose, CA, USA), mouse IgG1k FITC (BD Bioscience, cat# 555748), CTLA-4 PE (Abcam, cat# ab210254), and mouse IgG2a PE (Abcam, cat# ab91363). Samples were analyzed with BD LSR Flow cytometer using 488, 575 filters. The data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Flow cytometry data shown in the figures of this manuscript are representative of at least two independent experiments. Each cell treatment was coming from a single well.