Mtt salt

The metabolic activity of biofilm was determined by the reduction of MTT assay according to the protocol of Denizot and Lang (1986) (link) with some modifications. Overnight grown cultures of V. cholerae strains were diluted 100 times and inoculated in 200 μl of fresh LB broth contained in 96-well flat-bottom polystyrene plates in the presence or absence of quercetin and naringenin Half-MBIC concentrations (COSTAR; Sigma). After 24 h of incubation at 37°C under static conditions, the culture was removed and washed thrice with water. The MTT salt (Sigma-Aldrich, United States) was dissolved in phosphate-buffered saline (PBS) to give a final concentration of 5 mg ml⁻¹. The culture medium was carefully removed, and the wells were washed five times with 0.85% (w/v) NaCl solution and air-dried. One hundred microliters of MTT solution were pipetted into each well and incubated for 3 h at 37°C. The insoluble purple formazan obtained by enzymatic hydrolysis of MTT by the dehydrogenase enzyme found in living cells was further dissolved in 100 μl of dimethyl sulphoxide (DMSO; Sigma-Aldrich, United States). The absorbance was then measured at 570 nm using a microplate reader (Nivo, Multimode).

For a thiazolyl blue tetrazolium blue (MTT)-based cell proliferation assay, cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at a density of 0.5×10⁴ cells/well and treated with 4-fold serially diluted drugs for 72 hr. Then, 10 μl (5 mg/ml) of MTT salt (Sigma) was added to the corresponding well, the cells were incubated at 37°C for an additional 4 hr, and the reaction was stopped by lysing the cells with 150 μl of DMSO for 10 min. The optical density was measured at 570 nm. Both the linear regression parameters and the IC₅₀ (the concentration of drug required to kill 50% of the cells) values with the no-drug control as the reference were calculated. The relative drug resistance was presented as the fold change in the IC₅₀ of the cell lines relative to the lowest IC₅₀.

To assess the metabolic activity of the biofilm formed, the modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay was used according to the study by Schillaci et al. (2008) (link). MTT salt (Sigma Aldrich, São Paulo, Brazil) was dissolved in 0.1 M PBS to give a final concentration of 5 mg/mL. Plates containing preformed biofilm were incubated with 100 μL of each CFSM for 24 h. Then, the medium was gently removed, the plates were air dried, and 100 μL of MTT solution was added to each well and incubated for 3 h at 37°C under sterile conditions. The insoluble purple formazan (obtained by enzymatic hydrolysis of MTT by the dehydrogenase enzyme found in living cells) was further dissolved in 100 μL of dimethyl sulphoxide (DMSO, Sigma Aldrich, Darmstadt, Germany). The absorbance was then measured at an optical density of 570 nm using a microplate reader (Multiskan Spectrum, Thermo Scientific). The MTT assay was performed as three independent experiments with n = 6 biofilms per CFSM.

Cell viability was monitored using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay similar to what has been previously described [32 (link)]. Briefly, cells were plated at density of 2000 cells per well in flat-bottom 96-well plates and allowed to adhere overnight. Ten wells per strain were infected with 100 µL of ZIKV diluted to the indicated MOI. At the specified time points, 10 µL of 5 mg/mL MTT salt (Sigma-Aldrich) in EMEM solution was added to each well and incubated for 4 h at 37 °C, after which 100 µL of 10% SDS in 0.01 M HCl was added per well and the plates were incubated overnight at 37 °C. Absorbance at 550 nm with a reference wavelength of 650 nm was read on a Spark 10M plate reader (Tecan, Männedorf, Switzerland). The average absorbance of 10 wells was used and viability experiments were carried out in triplicate. The data was expressed in % Cytopathicity, which was defined as: % Cytopathicity = 100% − ((Uninfected Absorbance − Infected Absorbance)/(Uninfected Absorbance) × 100%).

For the assessment of cellular viability the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay was performed using MTT salt (Sigma, Cat. no. M5655) resuspended at 1 mg/ml in sterile water. Cells were plated out in 96 well plates at 500 cells per well and co-transfected as previously described, then on day 3 20 μl of the 1 mg/ml MTT solution was added to each well and left to incubate at 37 °C for 2–4 h The media from each well was decanted and replaced with 100 μl of dimethyl sulfoxide (DMSO) until all the purple formazan product was dissolved and the plate was read at 570 nm. Absorbance readings were taken as a percentage of the mean value of non-treated cells.