Colorimetric xtt assay kit

The cells were seeded in 96-well tissue culture plates at a density of 500-1000 cells/well by sextuplicate. After incubating for 24 hr, cells were incubated in the presence or absence of calcitriol (1X10^-8 M) during 48 hr. Afterwards, culture medium was removed and incubations with E₂ (1X10^-8 M), as an ER agonist, or tamoxifen (1X10^-6 M) and ICI-182,780 (1X10^-6 M), as ER antagonists, or their combination were performed in the absence or presence of calcitriol. Plates were incubated at 37°C for 6 days and cell viability was determined by using the colorimetric XTT Assay Kit (Roche) according to manufacturer’s instructions. After 4 hr incubation, absorbance at 492 nm was measured in a microplate reader (BioTek, Winooski, VT, USA).

Breast cancer cell lines were seeded in 96-well culture plates at a density of 1000-1200 cells/well depending on the cell line by triplicate. Then, the cells were treated in the absence or presence of different concentrations of calcitriol, EB1089 (0.01-100 nM), IL-1β, and TNF-α (0.05-100 ng/ml) or the combination of calcitriol with TNF-α. In addition, the cells were incubated in the presence of anti-IL-1R1 and anti-TNFR1 alone or in combination with calcitriol or the cytokines during 6 days at 37°C, 95% air, and 5% CO₂ in a humid environment. After incubation, cell proliferation was determined using the colorimetric XTT Assay Kit (Roche), according to the manufacturer's instructions. Absorbance at 492 nm was measured in a microplate reader (BioTek, Winooski, VT, USA).