Epoch2 spectrophotometer

To examine SARS-CoV-2 RBD-specific antibodies in the mouse sera, indirect ELISAs were conducted as published before [9 (link)]. Briefly, plates were coated with 0.2 µg/well of SARS-CoV-2 RBD proteins from different variants. Mouse sera samples were 3-fold diluted from 1:200 to 1: 11,809,800 in 0.1% BSA in an assay buffer (0.05% Tween20 in 1x PBS). Samples were prepared and measured in duplicate. Assay controls included a 1:400 dilution of pooled naïve mouse sera (negative control), 1:10,000 dilution of pooled high titer mouse (positive control), and assay buffer as blanks. A total of 100 µL/well of 1:6000 goat anti-mouse IgG HRP in assay buffer was added. After incubation, plates were washed five times, followed by adding 100 µL/well of TMB substrate. Plates were incubated for 15 min at room temperature (RT) while protected from light. After incubation, the reaction was stopped by adding 100 µL/well of 1 M of HCl. The absorbance at a wavelength of 450 nm was measured using a BioTek Epoch 2 spectrophotometer. For each sample, the titer was determined using a four-parameter logistic regression curve of the absorbance values. The titer cutoff value: negative serum control + 3 x standard deviation of the negative serum control.

100 µL aliquots of deep-frozen P. salmonis wild-type and mutant strains were streaked onto PSA plates and incubated for 2 days at 18°C. By this time, a homogeneous bacterial lawn had formed and we proceeded to suspend a loopful of bacteria in 1 mL of buffered saline solution (BSS: 8.5 g/L NaCl, 2.0 g/L K₂HPO₄, 2.0 g/L KH₂PO₄, pH 6.4), transferred them onto new PSA plates, and incubated as mentioned above. Bacterial suspensions (OD₆₀₀ = 1.0) were prepared by mixing bacteria with 5 mL of cold BSS in a conical tube kept at 10°C. For growth kinetics, 96-well microplates were inoculated with 150 µL/well of 1:100 bacterial suspensions in PSB and incubated during 70 h. Hourly reads were realized with an EPOCH2 spectrophotometer (Biotek, USA). By contrast, self-agglutination tests lasted 8 h, but used bacterial suspensions prepared in PSB that were held in plastic cuvettes at 18°C and pH 7.2. Reads were collected each hour with a densitometer (Ultrospec 10, GE LifeSciences). In order to determine bacterial counts, aliquots of log₁₀ dilutions in PSB were seeded on PSA plates in triplicates and incubated at 18°C for one week. Acriflavine tests were carried out using fresh bacterial suspension in BSS mixed with identical volumes (10 µL) of 0.1% acriflavine on a glass slide.

Tyrosinase inhibition activity was evaluated using the method used by Peyrot et al. [43 (link)]. In a 96-well microplate, 60 µL of ammonium formate buffer (50 mM, pH 6.4) were mixed with 10 µL of inhibitor solution at different concentrations: 10,000, 5000, 1000, 500, 100, 50, 10, 5, 1 and 0.5 µM in DMSO. Then, 20 µL of tyrosine at 4.42 mM in ammonium formate buffer was added. After 10 s of shaking, 10 µL of mushroom tyrosinase (5000 U.mL⁻¹ in ammonium formate buffer) were added and the mixture was incubated for 10 min at 37 °C. The amount of dopachrome produced during incubation was determined by absorbance readings at 420 nm every 15 s in a microplate Biotek (Winooski, VT, USA), Epoch 2 spectrophotometer. IC₅₀ values, corresponding to the concentration required to inhibit 50% of tyrosinase activity, were obtained using GraphPad Prism^® software version 6.01. Kojic acid was used as a reference and all measurements were performed at least in duplicate.