Spectramax plus 384 microplate reader

Biofilm formation test was carried out according to Vaishampayan et al. (2018) (link). Briefly, bacterial isolates grown overnight in LB medium at 37°C with shaking (150 rpm) were diluted to an initial OD₆₀₀ of 0.05 and re-grown to exponential phase (OD₆₀₀ ∼1.5). Cultures were transferred to translucent 96-well microtitration plates (Carl Roth^® GmbH & Co. KG) in replicates of eight and incubated at 37°C for 24 h. After careful removal of planktonic cultures, the wells were washed twice with 12 mM phosphate buffered saline (pH 7.4) and dried at 55°C for 1 h. Biofilms were stained with 200 μL 0.4% crystal violet solution and briefly rinsed with demineralized water. Biofilm formation was measured at 570 nm (OD₅₇₀) in a SpectraMax^® 384 Plus Microplate Reader (Molecular Devices). Staphylococcus aureus 04-02981, a strong biofilm former, was used as a positive control, LB medium was used as negative control. Means of five values each and three biological replicates were used. The following criteria were used for the interpretation of the results, ODc = negative control; OD ≤ ODc = non-adherent, ODc ≤ OD ≤ (2 × ODc) = weakly adherent, (2 × ODc) < OD ≤ (4 × ODc) = moderately adherent, (4 × ODc) < OD = strongly adherent (Nyenje et al., 2013 (link)).

The optical transmissions of the hydrogels were examined by a UV-Vis spectrometer (Molecular Devices SpectraMax 384 Plus Microplate Reader, Molecular Devices; San Jose, CA, USA). Six mm diameter trephined discs of the hydrogels were placed in individual wells of a 96-well quartz microplate, and their optical transmittance was recorded from 200–800 nm in quartz microplate at 1 nm wavelength increments. The transmittance of the samples was corrected with blank water media and the mean transmittance (%) for each group was calculated and plotted as a function of wavelength.

Human follicular fluid (FF) and serum were requested and retrieved from the CReATe Fertility biobank and Taipei Medical University Hospital and thawed on ice. FF (100ul) was cleared by centrifugation to remove any precipitate or cell debris (15 min 3,000xg, 4C), and FF and serum were assayed in duplicate for NPY using NPY ELISA kit, according to the manufacturer’s protocol (EMD Millipore Corporation). Absorbance was read at 450 and 650 nm with SpectraMax® 384 Plus microplate reader (Molecular devices, San Jose, CA) and concentrations were calculated using linear regression. The sensitivity of the assay was 3.9 pg/ml, intra and inter assay variabilities were 2.07 ± 0.62% and 0.92 ± 0.78% (mean ± SD), respectively.

HEM cells were seeded in a 60 mm culture dish at a density of 5 × 10⁵ cells per well. 24 h after UVB irradiation or SM treatment, HEM cells were washed with PBS and then were solubilized with 1% Triton X-100 at room temperature for 15 min. The lysates were centrifuged at 12,000 rpm for 10 min to obtain a supernatant. After quantification, 100 μl of lysis buffer was transferred into the 96-well plate with 100 μl of 1 mM L-DOPA added to each well. After incubation at 37°C for 1 h, absorbance was measured at 490 nm with the Spectra Max 384 PLUS microplate reader (Molecular Devices, Sunnyvale, CA, United States).