Celltiter blue cell viability assay kit

Cell culture 96-well plates and flasks were obtained from Sarstedt (Nürnbrecht, Germany). Cell culture media (Minimal Essential Medium (MEM) and Dulbecco’s Modified Eagle Medium/F12 (DMEM/F-12) without phenol red) and supplements (fetal bovine serum (FBS), charcoal-stripped FBS (CD-FBS), L-glutamine and penicillin–streptomycin (P/S)) were purchased from Gibco, Thermo Fisher Scientific, (Waltham/MA, USA). Zearalenone (ZEN), α-zearalenol (α-ZEL), α-zearalanol (α-ZAL), 17-β-estradiol (E2), 4-nitrophenylphosphate, diethanolamine, magnesium chloride and sulforhodamine B (SRB) were obtained from Sigma Aldrich Chemie GmbH (Schnelldorf, Germany), whereas daidzein (DAI), equol (EQ), genistein (GEN) and glycitein (GLY) were purchased from Extrasynthese (Genay Cedex, France). Dimethly sulfoxide (DMSO), NaCl, KCl, Na₂HPO₄, Na₂HPO₄ × 2 H₂O and KH₂PO₄ were purchased from Roth (Karlsruhe, Germany). ZEN-4-sulfate ammonium salt was obtained from Santa Cruz Biotechnology (Dallas/TX, USA) and is the same compound as ZEN-14-sulfate (ZEN-14-S) using the newer International Union of Pure and Applied Chemistry (IUPAC) numbering system (Metzler 2011 (link)). The CellTiter-Blue® Cell Viability Assay Kit was purchased from Promega Corporation (Madison/WI, USA).

The CellTiter-Blue cell viability assay kit (Promega) was used to count viable cells. 72 hours after virus infection, cells were seeded in 96 well plates at a density of 1000 cells per well in growth media. Twenty microliters of CellTiter-Blue reagent was added to 100 µl medium and incubated for 1 hour at 37 °C incubator. Fluorescence (560 nmEx/590 nmEm) was continuously measured for 5 days using TECAN plate reader. For drug treatment, cells were cultured in the growth media with 2% heat inactived FBS with different doses of trametinib (Skellchem) for 5 days, DMSO was used as vehicle control. Relative cell numbers were converted with standard curve of each cell line.

DNA synthesis was measured using ³H thymidine incorporation as previously described [8 (link)], or using an ELISA BrdU labeling kit (Catalog #11647229001, Roche Diagnostics, Indianapolis, IN) following manufacturer's protocol. Cell viability was measured using the CellTiter-Blue^® Cell Viability assay kit (Catalog # G8080, Promega, Madison, WI) as instructed by the manufacturer.

Cell adhesion was evaluated with the CellTiter-Blue Cell Viability Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, RBM cells were seeded on samples at a density of 4 × 10⁴/cm² and allowed to attach for 1, 3, 6, and 24 h. At predetermined time points, non-adherent cells were removed by rinsing with PBS. CellTiter-Blue Reagent (50 μL) and PBS (250 μL) were then added to each well. After incubation at 37 °C for 1 h, the solution was removed from the tissue culture plates and 100 μL was transferred to a new Falcon 96-well tissue culture plate (BD Biosciences). The optical densities at 560 and 590 nm of the remaining solution were measured. The difference between the two optical densities was defined as the proliferation value.

DLD1 and HCT116 cells previously transduced with the appropriate lentiviral constructs were seeded in 96-well plates at a density of 2 × 10³ cells in 50 µl of DMEM-FBS and co-transduced with additional 50 μl of 0.45 µm filtered supernatant from the corresponding gRNA Lenti-X™ 293 T transduced cells. Cell viability was interrogated at the indicated timepoint using the CellTiter-Blue Cell Viability Assay kit (Promega, #G808B) according to the manufacturer’s instructions. Fluorescence measurements were obtained using a 530(25) excitation and 590(35) emission filter with the automated microtiter plate reader Synergy HT (BioTek). Well-to-well variability was corrected for using a row-wise normalization approach for proper statistical calculations.