M200 pro

Total phenolic content (TPC) of the huangqin water and ethanol extracts was determined according to laboratory protocol. A mixture of 3 mL of ultrapure water, 50 µL of solvent, standard gallic acid or huangqin extraction sample, and 250 µL of FC reagent were vortexed for 5 s in a test tube. After a few minutes, 750 µL of 20% Na₂CO₃ (w/v) was added and a two-hour reaction was started in the dark at room temperature. The absorbance of all samples was measured at 765 nm by multifunction microplate reader (Tecan M200 Pro, Tecan Group Ltd., Mannedorf, Switzerland). The TPC of the huangqin water and ethanol extracts was showed as milligram gallic acid equivalents per gram of huangqin sample (mg GAE/g).

To determine whether WSSV infection affects the concentration of calcium ions, we used the intestine tissue of shrimp to detect the free calcium ions using a Calcium Colorimetric Assay Kit (Beyotime Biotechnology, Shanghai, China). Shrimp intestines were collected at 0, 10, 20, and 30 min, and 1, 2, 6, and 12 h post WSSV challenge for calcium detection. The intestine tissue (30 mg) was homogenized in 300 μL of sample lysis buffer using a glass homogenizer, followed by centrifugation at 14,000 × g for 5 min. First, the protein concentration of the supernatant was measured using the Bradford method. The supernatant was placed on ice for the subsequent measurement. Next, the calcium standard solution was diluted to different concentrations to create a standard curve. The concentration of the supernatant was detected following manufacture’ protocols using a microplate reader (TECAN M200 PRO, Tecan Group Ltd., Männedorf, Switzerland) at 575 nm.

The pseudovirion neutralization assay (PsVNA) for detection of neutralizing antibodies in sera uses a replication-restricted, recombinant vesicular stomatitis virus (rVSV*ΔG) expressing luciferase, which is pseudotyped with the VEEV IAB E1/E2 glycoproteins (TrD)^{38 (link)}. Briefly, heat-inactivated NHP sera (56 °C for 30 min) was first diluted 1:20, followed by five-fold serial dilutions that were mixed with an equal volume of Eagle’s minimum essential medium with Earle’s salts and 10% FBS containing 4000 fluorescent focus units of VEEV E1/E2 pseudovirions. This mixture was incubated overnight at 4 °C. Following this incubation, 50 μL was inoculated onto Vero 76 (ATCC, Manassas, VA) cell monolayers in a clear bottom, black-walled 96-well plate in duplicate. Plates were incubated at 37 °C for 18–24 h. The media was discarded and cells were lysed according to the luciferase kit protocol (Promega, Madison, WI). A Tecan M200 Pro (Tecan Group Ltd) was used to acquire luciferase data. The values were graphed using GraphPad Prism 9 software and used to calculate the percent neutralization normalized to cells alone and pseudovirions alone as the minimum and maximum signals, respectively. The percent neutralization values for duplicate serial dilutions were plotted. Eighty percent PsVNA (PsVNA₈₀) titers were interpolated from 4-parameter curves, and geometric mean titers were calculated.