Vybrant mtt cell proliferation kit

THP-1 cell-derived macrophages in 96-well plates were infected with only H. pylori or infected simultaneously with Lactobacillus strains for 8 h. Unbound bacteria were removed by washing the cells three times. Cells were then incubated with cell culture medium containing 100 mg/ml gentamicin for 2 h. After gentamicin treatment, the supernatants were spread on plates to confirm that all extracellular bacteria were dead. The cells were washed again to remove the antibiotics, and the cell viability was evaluated using a Vybrant® MTT Cell Proliferation kit (Thermo Fisher Scientific), following the manufacturer's instructions. The viability of cells was given as the percentage relative to unstimulated cells.

Proliferation was evaluated using both Alamarblue^® kit (USA, Thermofisher—cat DAL1025) and Vybrant^® MTT cell proliferation kit (USA, Thermofisher—cat V13154) according to the protocols provided by the manufacturers. Briefly, LAPC4 and prostate-induced bone metastasis cells were seeded at a density of 5000 cells/well in 96 well plates (USA, Costar, FisherScientific—cat 3882) coated with poly-l-lysine (USA, Sigma—cat P4707-50ML) and were grown in standard conditions (RPMI, 10% FBS, 1% PS) for 24 h. The next day, cells were treated with vehicle (PBS1x) or zol (USA, Sigma—cat SML0223-50MG) in low-serum conditions (1% FBS) for 7 days. The media was replaced (with either drug or vehicle) on day 4 for each experiment. For alamarblue^® assay, almarBlue dye was added to media at 1:10 dilution on day 7 and cells were incubated at 37 °C for 4 h. For Vybrant^® MTT cell proliferation assay, the cells were labelled with MTT at 1:10 dilution on day 7 and incubated for 4 h at 37 °C. Then, 75 µl of media containing MTT was removed from each well before adding 50 µl of DMSO (USA, Sigma– cat D2438) for each well and incubating cells for 10 min at 37 °C. After incubation, fluorescence of alamarblue (Excitation—540 nm, Emission 585) or the absorbance of MTT (540 nm) was analyzed using the Infinite Tecan M200 Pro microplate reader (Tecan Trading AG, Männedorf, Switzerland).

Cancer cells were seeded at a density of 3000 (HCC827) and 5000 (lung cancer-induced bone metastasis) cells per well in Costar 96 well plates (Fisher Scientific—cat 3882, Canada) with triplicate wells per tested variable in media supplemented with 10% FBS. After 24 h, fresh media with variables were added [vehicle (PBS1x) or zoledronate at concentrations 1, 3 or 10 µM (Sigma—cat SML0223, USA) in low serum-RPMI (1% FBS, 1% PS)]. Experiments were conducted for 7 days with a media change and variable replenishment on day 4. Two metabolic activity-based assays were used to assess proliferation: alamarBlue® (Thermofisher—cat DAL1025, USA) and Vybrant® MTT cell proliferation kit (Thermofisher—cat V13154, USA) as previously described [61 (link)]. For both assays, the plate was read using the Tecan Infinite M200 Pro plate reader (Tecan Trading, AG, Männedorf, Zürich, Switzerland) with the following setting: for alamar (excitation wavelength 540 nm and emission wavelength 585 nm) and MTT (absorbance wavelength of 540 nm). Raw data generated from both assays in excel sheets were after background-normalized and analyzed as ratio of zoledronate-treated values over vehicle-treaded values.