384 well microplate

The 2-D cultures of G. neptuni cells were incubated in M1 medium to create low density (5 × 10⁶ cells/mL) and high density (1 × 10⁷ cells/mL) controls. In total, 30 µL of each inoculation cell density were added in triplicate to a 384-well microplate (Corning, Corning, NY, USA). The plates were then sealed and incubated at room temperature in the dark for 21 days. Monitoring occurred as described above. Cells were counted by pipetting the contents of each sacrificed well to resuspend the cells and then the cells were counted using an automated cell counter. As total medium replacement resulted in a loss of sponge cells, half medium exchanges were made at 12, 24, and 48 h, and every 48 h thereafter.

ABTS was used as a substrate for RbMPO to measure peroxidase activity. Measurements were performed in a 384-well plate (Corning^® 384 well microplate) at 37 °C and each condition was performed in triplicate. Fixed concentrations of RbMPO and H₂O₂ were used (0.5 nM and 79 µM, respectively) in a final volume of 20 µL in 50 mM sodium phosphate buffer pH 5.5. The plate reader was configured to run a 10 min kinetics at 420 nm (ε_ABTS.₊ = 36 mM⁻¹·cm⁻¹, see Table 1) with the following ABTS range: 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30 and 40 mM.

The nine probes, including a positive control, a negative control and 7 virus-specific oligonucleotides probes described in Table 2, were diluted to 50 μM with DNA spotting buffer (CapticalBio Corporation, Beijing, China) following the manufacture’s instruction. The probes were then added into a 384-well microplate (Corning Life Sciences, Tewksbury, MA, USA) and spotted on aldehyde coated glass slides (Shanghai Biotech Co., Shanghai, China) with a contact microspotting robotic system (OminiGrid 100 Arrayer, GeneMachines, San Carlos, CA, USA). Each slide contained 12 identical arrays on which the nine probes for 7 s viruses, a positive control and a negative control (Table 2) were arranged. Each probe was printed seven replicates dots with 300 μm of spacing and 100 μm of diameter. Each individual line of seven dots from number 1 to 7 represented one specific virus. The positive control probe was spotted on the top and bottom line while the negative control was displayed on the penultimate line. The patterns of the viral array with nine probes were indicated in Fig. 1.The relative humidity and temperature of spotting environment were about 70% and 25°C. The spotted slides were stored in drying cabinet until use.

DNA-binding reactions (20 μl) were performed at 25 °C in buffer containing 25 mM HEPES pH 8.0, 0.2 M KOAc, 10% glycerol, 0.25 mM TCEP, 1 mM Mg(OAc)₂ and 0.01% Brij-35. Proteins were serially diluted and mixed with 10 nM (final concentration) of FAM-labelled DNA in 384-well microplates (Corning). The plates were measured using the CLARIOstar microplate reader (BMG Labtech). Blank-corrected anisotropy measurements were averaged and plotted against protein concentration. RAD52 binding was curve-fitted using the following quadratic equation in GraphPad Prism 9 to determine K_D values: $$Y=A_{\min }+\left(A_{\max }{-A}_{\min }\right)\times \frac{x+L+K_{\mathrm{D}}-\sqrt{{\left(x+L+K_{\mathrm{D}}\right)}^2-4\times x\times L}}{2\times L},$$ where Y is the fluorescence anisotropy, A_min and A_max are the minimum and maximum fluorescence anisotropy values, L is the ligand concentration (equal to 0.01 µM), x is the protein concentration and K_D is the dissociation constant. At least three independent triplicates of technical replicates were performed for each binding condition.