Biotek synergy ht plate reader

The ORAC assay was based on the procedure previously described by Kőszegi et al. (2017) and Patay et al. (2016) [53 (link),54 (link)] without modifications. In summary, a fluorescein working solution (400 nmol L⁻¹, Merck Life Science Ltd., Budapest, Hungary) and the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) oxidant (400 mmol L⁻¹, Merck Life Science Ltd., Budapest, Hungary) dissolved in 75 mmol L⁻¹ potassium phosphate buffer (mixture of KH₂PO₄ and K₂HPO_4, Reanal Labor, Budapest, Hungary) at pH 7.5 were prepared freshly before the measurements. Trolox standards were prepared in the potassium phosphate buffer (0–160 µmol L⁻¹). Into each well, 25 µL of the blank/standard/sample and 150 µL of fluorescein solution were added in optical plates (Perkin Elmer) and the mixture was incubated at 37 °C for 30 min in the dark. Next, 25 µL AAPH solution/well was injected by the automated injector of a Biotek Synergy HT plate reader (BioTek Instruments, Winooski, VT, USA) previously warmed up to 37 °C. The fluorescence intensities were monitored for 80 min (490/520 nm wavelengths) at 2 min intervals. The area under each curve (AUC) was obtained using the software of the reader providing the total sum of the individual digital data of the corresponding fluorescence signals. The antioxidant capacity values were expressed as µmol Trolox equivalent (TE) g⁻¹ honey.

The NO production was measured based on the amount of nitrite accumulated in the culture supernatants using the Griess assay. This consists of a two-step diazotization reaction in which the NO derived nitrosating agent, dinitrogen trioxide (N₂O₃), generated from the acid-catalyzed formation of nitrous acid from nitrite (or autoxidation of NO), reacts with sulfanilamide to produce a diazonium ion, which is then coupled to N-(1-napthyl)ethylenediamine to form a chromophoric azo product that absorbs strongly at 540 nm [39 (link)]. Equal volumes of the culture supernatants and reagents (equal volumes of 1% (w/v) sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) N-(1-napthtyl) ethylenediamine dihydrochloride) were mixed and incubated for 10 min at room temperature and placed in the dark. The concentration of accumulated nitrite in the culture supernatants was calculated by interpolation of the absorbance of each sample, read in a Biotek Synergy HT plate reader (Biotek) at 550 nm, in a standard curve of sodium nitrite.

Free glycogen in genital fluid samples was measured fluorometrically using the Glycogen Assay Kit (BioVision, Milpitas, CA). Briefly, 10 μl of genital fluid or saline (serving as blank) was added to test wells in a 96-well plate. The volume was adjusted to 50 μl with hydrolysis buffer, with (total) or without (glucose background) the kit hydrolysis enzyme. Glucose background (samples without hydrolysis enzyme) was subtracted to determine glycogen concentration. The Relative Fluorescence Units (RFU) were measured using a BioTek Synergy HT plate reader (BioTek Instruments, Inc, Winooski, VT). Samples were diluted in 1X PBS (1:25, 1:50, 1:200, and 1:400) and glycogen was measured for each dilution in duplicate assays. The intra-assay coefficient of variation (CV) was between 10–13% All samples from one subject were run in one assay to avoid batch-to-batch changes in the assay.

The ability of strains to produce biofilms was assessed using a method adapted from Anderson et al. [105 (link)]. Non-piliated bacteria, cultured as above, were collected from plates and suspended to OD₅₅₀ = 1.5 in supplemented GCBL. One hundred microliters of this suspension were added to the wells of a 96-well flat-bottomed microtiter plate (Corning #3370). Water was added to all remaining wells to minimize the effects of evaporation. The plate was wrapped in plastic wrap and incubated at 37 °C in a 5% CO₂ environment for 24 h without shaking. After incubation, samples from each well were serially diluted and spotted onto GCB plates for CFU/mL enumeration of viable planktonic bacteria. Planktonic bacteria and spent media were removed and biofilm wells were washed once with PBS. Plates were allowed to dry for ~4 h at room temperature, after which 65 μL of 0.1% crystal violet were added to each well and incubated for 15 min at room temperature. Wells were then washed 3 times with PBS and allowed to dry overnight at room temperature. Biofilms were dissolved by the addition of 125 μL 30% acetic acid, incubation at room temperature for 30 min, and shaking on a microplate vortexer. Biofilm mass was measured on a BioTek Synergy HT plate reader (BioTek) at 550 nm.