Celltiter 96 aqueous one solution cell kit

Cell viability was assessed using the CellTiter 96 AQueous One Solution cell kit (Promega), which contains a tetrazolium compound MTS. Cl.8+ cells were seeded at a concentration of 1 × 10⁶/ml in a flat-bottomed 96-well plate in minimal medium (MM) with or without recombinant IDGF2 (16 μg/ml), Ado and dAdo and incubated for 22 h. Next, 20 μl of MTS solution was added to each well and the plate was incubated for an additional 3 h at 25 °C. The absorbance at 490 nm was recorded with a 96-well plate reader. The assay was performed with five replicates for each sample.
Proliferation of Cl.8+ cells in SFM and CM was also measured by the direct counting of cells using digital photographs of identical areas (0.8 × 0.8 mm) taken every 24 hrs. Each value represents an average of three fields per plate.
TUNEL staining, BrdU incorporation, the ATP assay and the Ado uptake assay were performed as described previously20 (link). For TUNEL staining, we used the In Situ Cell Death Detection kit with fluorescein (Roche), and for BrdU incorporation, we used the In Situ Cell Proliferation Kit, FLUOS (Roche). ATP was measured by The CellTiter-Glo^®Luminescent Cell Viability Assay (Promega). The Ado uptake assay was performed using H³-Ado in MM.

We followed a previous method [17 (link)]. The MTS assay was conducted as the cell viability assay in this study. Breast cancer cells (10⁴ cells/well) were cultured in a 96-well plate for 24 h. The breast cancer cell lines were treated with increasing concentrations of ciclesonide (0, 5, 10, 20, 40, and 80 µM) for 1 day. Cell proliferation was assessed using a CellTiter 96^® Aqueous One Solution cell kit (Promega, Madison, WI, USA). After mixing DMEM and aqueous one solution (5:1), we added 100 µL of mixture to each well and incubated the cells at 37 °C for 1 h and the OD₄₉₀ was measured using a microplate reader (SpectraMax, San Jose, CA, USA).