Microwell plate

The experimental setup for X-ray irradiation consists of a system aligned on a vertical axis, with variable source-to-object distance in the range of 90–375 mm. Polystyrene Petri dishes or microwell plates (Corning/Costar Inc., Cambridge, USA) are placed above a 1 cm-thick block of polymethyl methacrylate (PMMA) [15 (link)]. For operator safety, the whole system is enclosed in a shielded cabinet (<1 μSv/h at each accessible point during operation). The X-ray source (Apogee, Oxford Instruments, USA) is a packaged X-ray tube with fixed tungsten anode and 125 μm-thick beryllium window, operating in continuous mode. An additional 1 mm-thick aluminium filter was placed in front of the X-ray exit window. The maximum accelerating voltage and filament current are 50 kV and 1 mA, respectively (50 W max. continuous power).
The X-ray tube is operated by a general purpose personal computer through a dedicated controller with RS232 interface (ADIO232, RFS Systems, Straubenhardt, Germany). The average irradiation time for a total dose of 0.25 Gy was 164 s, corresponding to a dose rate of 91 mGy/min.
For all the experiments, the irradiation parameters were 50 kV, 0.7 mA, source-to-object distance = 211 mm. Thermoluminescent dosimeters (TLD-100, Harshaw) at the same position of the specimen were used before experiments to calibrate the system in term of absorbed dose.

Nitric oxide production by macrophages was assayed by determining the increase in nitrite concentration [23 (link)] by the Griess reaction adapted to microwell plates (Costar). Briefly, 50 μL of culture supernatant was mixed with an equal volume of Griess reagent and incubated for 10 min at room temperature in the dark; the absorbance was measured at 570 nm in an automatic microplate reader (Organon Technika Microwell System). Values were quantified using serial dilutions of sodium nitrite.

The binding epitopes of the MAbs were analysed using a competition ELISA with rWNV-NS1 protein as the coating antigen. Microwell plates (Costar Corning, Inc., Corning, NY, USA) were coated with 100 µl/well of rWNV-NS1 at a concentration of 1 µg/ml. After blocking, non-HRP-conjugated MAb at a concentration of 500 µg/ml was incubated with HRP-conjugated MAb at a dilution of 1∶500 at 37°C for 1 hr. After the plates were washed, colour reactions were developed using tetramethylbenzidine (TMB, KPL, Gaithersburg, VA, USA) with hydrogen peroxide (H₂O₂) as a substrate solution and stopped with 0.3 N sulphuric acid (100 µl/well); the absorbance was read at 450 nm in an ELISA plate reader (Bio-Tek, Winooski, VT, USA). The influenza virus NS1 protein MAb IVNS1-M6 [33] was used as an irrelevant control. The percentage of inhibition was calculated according to the following formula: [1-(OD₄₅₀ of the test well/OD₄₅₀ of the control well)] ×100%, where OD₄₅₀ is the optical density at a wavelength of 450 nm. If the inhibition were greater than 75%, it was defined as competitive inhibition; if the inhibition were between 75% and 25%, it was defined as relative competition; and if the inhibition were <25%, it was defined as non-competitive inhibition.

Cells were seeded and incubated at 5% CO2, 37°C overnight. Different concentrations of test compounds were added and incubated for another 2 h at 5% CO2, 37°C. Whole-cell lysates of HT-29 cells were prepared by RIPA buffer, and subjected to ELISA analysis.
Microwell plates (COSTAR) were coated with capture antibody in coating buffer (100 µL/well). After overnight incubation at 4°C, the coated wells were washed three times with washing buffer and blocked at 37°C for 1 h by adding 100 µL of 2% BSA diluted in 0.1% Tween 20 in PBS. After a further washing procedure, 100 µL of standard dilutions and samples were added and the plate incubated for 1 h at 37°C. After washing three times, HRP-conjugated secondary antibody was added to the wells and the plates were incubated for 1 h exactly. After a final washing step bound antibodies were detected by the addition of 100 µL of TMB (Thermo 1-Step Ultra TMB). After 15 min the reaction was stopped with 100 µL 2 mol/L H₂SO₄ (sulphuric acid). Optical density (OD) was measured at 450 nm within 30 minutes.

The silver nitrate (AgNO₃, 99.8%) was purchased from Sinoreagent (Shanghai, China). Tween-20 and Chloroauric acid (HAuCl₄) were obtained from Amresco (USA). Trisodium citrate and 4-aminothiophenol (4-ATP) were obtained from Sigma-Aldrich. Magnetic beads (MBs)@Streptavidin was purchased from BEAVER (Suzhou, China). The RNase Inhibitor and the NEBuffer 2.1 were obtained from the New England Biolabs (USA). Microwell plate was obtained from CORNING, USA.