Cell Proliferation Assay for Pancreatic Cancer

The proliferation of PC cells was evaluated with a Cell Counting Kit-8 (C0038, Beyotime) following the manufacturer’s instruction. Briefly, 5x10⁴ Capan-1 and PANC-1 cells transfected with B7H6-siRNA or scramble siRNA were seeded separately into wells of a 96-well plate and cultured in 100 μL of cell culture media. At the indicated time points, the media were replaced with CCK-8 reagent (10 μL CCK-8 and 90 μL Media), and the cells were incubated for an additional hour. Absorbance of each well was then measured at 450 nm to determine cell growth, which was monitored every 24 hours over 3 days. For real time cell analysis (ACEA Bioscience, xCELLigence RTCA MP), as reported earlier (28 (link)), PC cells were seeded at a density of 5,000 cells/well and incubated in an xCELLigence RTCA MP instrument at 37°C for 72 hours. Data were collected every 15 minutes for a total of 72 hours. The cells’ normalized cell index was analyzed using xIMT software (ACEA Biosciences Version 2.3.2). Cells were also seeded into 6-well plates at 1×10⁵ cells per well and incubated for 72 hours so that cell morphology could be observed with a microscope.