Labsystems multiskan ms plate reader

Proliferative Caco-2 cells in monoculture were detected using the 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT) assay, according to the ISO 10993-5 International Standard procedure (ISO 10993-5, 2009). The method is based on the reduction of MTT by mitochondrial dehydrogenase of intact cells to produce purple formazan, determined by measuring the absorbance at 540 nm using a multiwell scanning spectrophotometer (Labsystems Multiskan MS Plate Reader, ThermoFisher Scientific), as described by Truzzi et al. [53 (link)].

Quantitative biofilm formation assays were carried out as in our previous work [5 (link)]. Overnight EAEC cultures, grown in LB medium, were sub-cultured (1 in 100) into 5 ml of DMEM (Dulbecco’s Modified Eagle Medium) high glucose (Sigma) and incubated at 37°C for 1 hour with shaking. 150 µl of each culture was pipetted into a microtiter plate in triplicate, which was sealed with a Breath Easy gas permeable membrane (Sigma), and then incubated overnight statically at 37°C. After ~16-17 hrs the spent media was removed and 150 µl of 0.1% (w/v) crystal violet was added to each well and left at 4°C for 30 minutes. The crystal violet solution was removed, the plate washed thoroughly with water, excess liquid removed and 150 µl of ethanol/acetone solution (80 ml ethanol and 20 ml acetone) was added. The plate was left on a shaker for 30 minutes at room temperature and the absorbance was measured at 595 nm by a Labsystems Multiskan MS plate reader (Thermo Fisher Scientific Inc).

Proliferative Caco-2 and NCM460 cells in monoculture were detected using the MTT assay, according to the ISO 10993-5 International Standard procedure (ISO 10993-5, 2009). The method is based on the reduction of MTT by mitochondrial dehydrogenase of intact cells to produce purple formazan, determined by measuring the absorbance at 540 nm using a multiwell scanning spectrophotometer (Labsystems Multiskan MS Plate Reader, ThermoFisher Scientific, Waltham, MA, USA), as described by Truzzi et al. [22 (link)]. Results were expressed as the percentage of viable cells with respect to untreated controls (70% ethanol). The percentage of cell proliferation was calculated using the following formula: absorbance value of treated sample/absorbance value of control × 100 = % of cell viability.