Powerwave xs microplate spectrophotometer

Liberated benzyl mercaptan was labelled as follows, 500 μl from each reaction was taken at the appropriate time point and centrifuged at 13 000 r.p.m. for 2 min. To chemically label benzyl-mercaptan, 50 µl of labelling reagent, consisting of 4.09 mM 3-(4,5-dimethylthiazol-2-yl)−5- (3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium (MTS) (Promega) and 0.2 mM phenazine methosulfate (PMS) (Sigma) (Goodwin et al., 1995 (link)), dissolved in dH₂O, was aliquoted into either a 96 well microplate or 0.5 ml Eppendorf tubes. For reactions using a 96 well microplate, 200 µl reaction supernatant was added to the pre- aliquoted 50 µl MTS/PMS. The plate was sealed with a semi-permeable film lid, incubated at room temperature for 50 min and the A_492nm recorded on a Biotek PowerWave XS microplate spectrophotometer. The data was captured by KC junior and exported to Microsoft Excel. For reactions in Eppendorf tubes, 20 µl - 50 µl reaction supernatant was added to 50 µl MTS/PMS. The reaction was incubated at room temperature for 50 min. 20 µl was subsequently removed and diluted in 980 µl dH₂O in a 1.5 ml disposable plastic cuvette. The A_492nm was recorded.

The spectrophotometric assay was performed in 96-well plates. Plasma (80 µL) was centrifuged at 10,000 g for 30 min at room temperature and subsequently mixed with 60 µL of 5 mM hydrogen phosphate and 60 µL of 41.67 mM calcium to attain final concentrations of 1.5 mM phosphate and 12.5 mM calcium, respectively. All reagent solutions were filtered and pH adjusted to 7.4. Crystallization of calcium phosphate was monitored spectrophotometrically for 30 min at room temperature via increase in absorbance at 550 nm using the Biotek Powerwave XS Microplate spectrophotometer. The plate was incubated at room temperature in an orbital shaker (750 rpm) and absorbance measured every 3 min. Plasma crystallization potential was assessed based on slope measurement in the linear range between 6 and 24 min from plots of increase in absorbance versus logarithm of time.

For the growth curves, cultures were diluted to OD₆₀₀ 0.05 ± 0.005 in SD-glucose and SD-galactose and incubated at 30°C with orbital shaking for 24 h. Growth was monitored hourly by measuring OD₆₀₀ using a Biotek Power Wave XS Microplate Spectrophotometer (Biotek^®, Winooski, United States). Model-free spline (nonparametric) and model fitting (parametric) approaches were used to calculate the growth parameters in the R software (35 (link), 36 (link)). The package grofit (37 (link)) was used to adjust a model-free spline and the parameters maximum cell growth (μm) and length of the lag phase (lag time), were estimated from the spline fit. The same package was used to adjust to a model-based curve and the parameters were estimated from the best fit model. The 95% confidence intervals (CIs) were calculated via bootstrapping for both model-free spline and model-based fits. The results are represented by the best model curve with 95% CIs for each strain compared to the control strain (not expressing IAPP).

To assess the cytotoxic potential of CNPs we employed the MTT assay. Briefly, L929 cells were treated with samples in various concentrations of 500, 100, 50, 10, and 5 nM for 24 h at 37°C in a humidified 5% CO₂ incubator. After, cells were washed with PBS and 50 μL of MTT (2 mg/mL) was added followed by incubation for 4 h. The medium was removed and cells were washed followed by DMSO addition. Absorbance at 570 nm was determined (BioTek, PowerWave XS microplate spectrophotometer) and the percentage of viable cells was determined relative to the untreated control group.
To assess UVA phototoxicity, L929 cells were treated with 100, 50, 10, and 5 nM CNP for 24 h. After, the CNP solution was replaced with HBSS and cells were irradiated with UVA (30 J/cm²) and then incubated with serum-free DMEM for 24 h. After, cytotoxicity was detected by MTT assay.

The cytotoxicity of treated L929 cells was assessed using the neutral red assay [16 (link)]. The cultured cell suspension was dispensed into a 96-well plate (2.5 × 10⁴ cells/well) and incubated for 24 h. Postincubation, the media were replaced with serum-free DMEM containing different concentrations (7, 14, 21, 28, and 35 μM) of DHCA with a maximum of 0.6% (v/v) DMSO or by serum-free DMEM containing 0.6% DMSO and incubated for 24 h. The monolayers were washed after incubation with phosphate-buffered saline (PBS), and 200 μL of neutral red (Interlab, São Paulo, SP, BR) solution (40 μg/mL) was added. The supernatant was removed after 3 h, and the cells were fixed with an aqueous solution of 2% (v/v) formaldehyde and 1% (w/v) calcium chloride, and then 200 μL of a solution of 1% (v/v) acetic acid and 50% (v/v) ethanol was added. Absorbance was measured at 540 nm (BioTek, PowerWave XS microplate spectrophotometer) after 15 min at 25°C in the dark. The percentage of viable cells was calculated relative to untreated cells.