Multiskan fc microplate spectrophotometer

The modified microplate dilution method described by the Clinical and Laboratory Standards Institute (CLSI) was used to determine the minimum inhibitory concentration (MIC) of the lyophilized supernatant of S. albidoflavus Q (23 (link), 66 ). Briefly, stock solutions were prepared with the lyophilized supernatant in RPMI 1640 medium (Sigma-Aldrich). The yeasts were then grown in YPD liquid yeast medium at 37°C for 24 h. The pre-inoculum OD₆₂₀ = 0.6 was established before performing serial 1:1,000 dilutions to obtain the inoculum. Solutions of the supernatant or fluconazole (the inhibition control) were placed in microplates and incubated at 37°C for 24 h. The absorbance was read in a Thermo Scientific Multiskan FC microplate spectrophotometer at 620 nm (23 (link)).

Myeloperoxidase activity (MPO), an index of polymorphonuclear cell accumulation, was determined in the vaginal tissues, as previously described [26 (link)]. Vaginal tissues collected at the specified time were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mm potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000× g at 4 °C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mm) and 0.1 mm H₂O₂. The rate of change in absorbance was measured spectrophotometrically at 650 nm using a Thermo Scientific™ Multiskan FC Microplate Spectrophotometer (Model: 51119100). MPO activity was expressed in units/mg protein.

The modified microplate dilution method described by the Clinical and Laboratory Standards Institute (CLSI) was used to determine the minimum inhibitory concentration (MIC) of the lyophilized supernatant of S. albidoflavus Q (23 (link), 66 ). Briefly, stock solutions were prepared with the lyophilized supernatant in RPMI 1640 medium (Sigma-Aldrich). The yeasts were then grown in YPD liquid yeast medium at 37°C for 24 h. The pre-inoculum OD₆₂₀ = 0.6 was established before performing serial 1:1,000 dilutions to obtain the inoculum. Solutions of the supernatant or fluconazole (the inhibition control) were placed in microplates and incubated at 37°C for 24 h. The absorbance was read in a Thermo Scientific Multiskan FC microplate spectrophotometer at 620 nm (23 (link)).

The effect of 1c and 5b on the growth of C. glabrata CBS 138 and C. glabrata 43 was evaluated by using the CLSI M27-A3 microdilution method. Briefly, stock solutions of antifungal compounds were prepared, from which the experimental concentrations were obtained in RPMI 1640 medium (Sigma-Aldrich). Fluconazole, simvastatin, atorvastatin, and α-asarone served as reference compounds for examining susceptibility. C. albicans ATCC 10231 and C. krusei ATCC 14423 were the controls for sensitivity and resistance, respectively. The synthetic compounds were dissolved in DMSO immediately before being placed on the microplates and were subsequently incubated at 37°C for 24 h. To avoid an inhibitory effect by the solvent, its volume was less than 10% of the total volume. Growth was quantified by optical density in a Thermo Scientific Multiskan FC microplate spectrophotometer at 620 nm. The values of yeast growth are expressed as the averages of three independent assays.

A comprehensive set of Th17-related cytokines was evaluated by ELISA: (i) cytokines that promote Th17 differentiation in naive T cells (IL-1β, IL-6, IL-21, IL-23, and TGFβ1), and (ii) cytokines produced by Th17 cells (IL-17A, IL-21, IL-22) [69 (link),70 (link),71 (link),72 (link)] (Supplementary Figure S1). Additionally, we evaluated the Th1 hallmark cytokines IFNγ and TNFα and the T regulatory (Treg) hallmark cytokine IL-10. All cytokines except IL-22 were evaluated using ELISA Ready-Set-Go! kits (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. IL-22 was evaluated in a separate ELISA kit purchased from Antigenix America, Huntington Station, NY. All serum samples remained undiluted during the evaluation of cytokine levels except for the evaluation of TGFβ1 levels in which serum was diluted 1:5. In short, the ELISA plates were initially incubated with cytokine-specific capture antibody, followed by the addition of patient serum, and finally the addition of cytokine-specific detection antibody. The binding of the detection antibody was visualized by adding avidin-conjugated horseradish peroxidase and an enzyme-specific substrate. Specific cytokine quantities were measured using a Multiskan FC Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and compared to manufacturer-supplied cytokine standards (presented in pg/mL).