Celltiter aqueous non radioactive cell proliferation assay

B16 melanoma, 3LL, and MC38 cell lines were obtained from the American Type Culture Collection (ATCC). The murine ovarian surface epithelial cell lines MOSEC-ID8, IG10, and IF5 were obtained from Katherine Roby (University of Kansas Medical Center, Kansas City, Kansas, USA) and grown as described previously (49 (link)). All cell lines were confirmed to be free of mycoplasma contamination. Cells were propagated in DMEM supplemented with 10% FBS and L-glutamine. An in vivo–passaged ID8 cell line that could be reintroduced into naive mice was generated by injecting mice with 1 × 10⁵ ID8 cells and then collecting and centrifuging the resulting ascites fluid on approximately day 80 (50 (link)). For tumor cell–Mϕ coculture experiments, 5 × 10⁵ peritoneal Mϕ and tumor cells were mixed (1:1) and incubated in 6-well plates for 48 hours. In some experiments, tumor cells were seeded on Transwell inserts containing 3-μM pores (Costar). In some experiments, tumor cells were treated with the MAPK inhibitor PD98059 (50 μM; Cell Signaling Technology), and cell proliferation was quantified using the CellTiter Aqueous Non-Radioactive Cell Proliferation Assay (Promega).

Carbenoxolone disodium salt (CBX; Sigma) was used as a gap junction inhibitor. Human connexin-32 plasmid (Cx32), C-terminally tagged with mEmerald, was obtained from Addgene (plasmid #54054) [48 (link)] while pcDNA 3.1 mock plasmid was purchased from Invitrogen. EVOS fluorescent microscope was used to verify the expression of Cx32 in cells (Additional file 3: Figure S3). Co-cultures of GS-293 and FSHR-293 cells were transiently transfected with Cx32 plasmid to determine the effect of connexin overexpression on cAMP transfer.
For determining cell viability after CBX treatment, co-cultures of GS-293 and FSHR-293 were preincubated with different concentrations of CBX (25, 50, 75 and 100 μM) for 2h in assay medium. Assay medium was then discarded, followed by washing with PBS. Cell viability was then assessed by CellTiter AQueous non-radioactive cell proliferation assay (Promega) following manufacturer’s instructions. Briefly, cells were incubated in DMEM complete medium (without phenol red) containing MTS/PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate] solution for 2h (37°C, 5% CO₂). Absorbance was then read at 490 nm, which provides a measure of cellular viability. One-way analysis of variance (ANOVA) was used to determine statistical differences among different samples.