Microplate spectrophotometer

Viable cells were measured using Cell Counting Kit-8 (CCK-8, Dojindo). In summary, cells were seeded onto 96-well plates at a density of 1 × 10⁴ cells per well. 10 μL of CCK-8 reagent (in 100 μL of medium per well) were incubated for 1 h after indicated treatment and time. Absorbance was then taken at 450 nm using a microplate spectrophotometer (Fisher Scientific, USA). Cells (250 for 0 Gy or 450 for 4 Gy) were seeded on 6-well and 12-well plates and incubated for 14 days after relevant treatment. Cell colonies were dyed with methylene blue for 10 min. Pictures of adherent cells were taken using Bio-rad Gel Doc XR imaging system (Bio-Rad, USA).

Levels of MDA (Cat#: A003-1-2) and the activity of SOD (Cat#: A001-1-2), GSH-Px (Cat#: A005-1-2) and CAT (Cat#: A007-1-1) (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) in brain tissue were evaluated by respective kits according to the manufacturer’s instructions. MDA, SOD, GSH-Px and CAT were measured spectrophotometrically at a wavelength of 532 nm, 450 nm, 405 nm and 405 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Hemolysis assay was performed by adopting the protocol as described earlier (25 (link)). Briefly, different concentrations of EVs or other components were mixed with an equal volume of 2% RBC and incubated at 37°C for 60 min. After removing undissolved RBCs by centrifugation (1,500 rpm, 10 min), 100 µL of supernatant was removed to a 96-well plate. Absorbance of the released hemoglobin in the supernatants was determined at 450 nm using a microplate spectrophotometer (Thermo Fisher Scientific). PBS and Triton X-100 (0.1%) were used as negative and positive controls, respectively. Hemolysis rate (%) = (EV group OD450 − negative control OD450) / (positive control OD450 − negative control OD450) × 100%.

Anti-HEV antibodies were detected by enzyme-linked immunosorbent assay (ELISA) using HEV genotype 3 -derived VLP as an antigen. For the detection of antigen bound IgG, anti-pig IgG antibody-HRP (Bethyl Laboratories Inc. TX. USA) conjugate was applied as a secondary antibody. The serum samples were diluted 1:100 with PBS containing 0.05% Tween 20, and 10% of Block Ace (DS Pharma Promo Co. Ltd. Osaka, Japan), and incubated for 1 h at room temperature. After the secondary antibody reactions, 50 μl of TMB (3,3′,5,5′-tetramethylbenzidine) (Kirkegaard & Perry Laboratories Inc., Baltimore, MD, USA) was added, and after a 10-min incubation at room temperature, 50 μl of 2 M sulphuric acid was added to stop the reactions. The optical density (OD) value at 450 nm was measured by a microplate spectrophotometer (Thermo Fisher Scientific Inc. USA). The Index value was calculated from the OD value obtained by ELISA as follows.
$$\text{Index value}:\frac{\text{Sample}\mathrm{OD}-\text{Control}\mathrm{OD}}{\text{Positive}\mathrm{OD}-\text{Control}\mathrm{OD}}\mathrm{x}100\left(\%\right)$$
The cut off value was set at 0.295 by calculating the average value + 2 standard deviation of negative samples (n = 5). As a result, the sensitivity and a specificity for the test were 90.0% (95% CI: 68.30–98.77) and 91.67% (95% CI: 73.0–98.97) based on the test results of 20 experimentally infected and 24 Negative pigs.

The viability of HDFs was determined using a CellTiter 96* aqueous nonradioactive cell proliferation assay (MTS; Promega, Madison, WI, USA). In brief, 20 μL MTS solution was added to each well of a 96-well plate and incubated for 2 h in a humidified incubator at 37 °C containing 5% CO₂. The formazan product was quantitated by measuring the absorbance at 490 nm with a microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).

ESCC cells were seeded at an initial density of 4 × 10³ cells per well on 96-well plates, and the cell viability was determined using methyl thiazolyl tetrazolium (MTT) assays over 24 h to 96 h. Specifically, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 h at 37 °C. Further, 150 μL of dimethyl sulfoxide was added to each well replacing the previous medium and incubated for 10 min. The absorbance was recorded at 570 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).