Uquant microplate reader

For luciferase assay, cells were maintained in DMEM with 10% fetal bovine serum and seeded at 1 × 10⁵ cells per well in 12-well plates 1 day before the assay. Using Lipofectamine 2000 (Invitrogen), cells were administered with 100 ng of pGL3-FGF2 promoter WT, E1 Mut or E2 Mut, together with 20 ng of CMV/β-galactosidase plasmid to normalize transfection efficiency. After 24 h, cells were washed with PBS and permeabilized with Cell Culture Lysis Reagent (Promega). Luciferase activity was measured with a Turner Biosystems TD-20/20 luminometer (Turner BioSystems, Sunnyvale, CA, USA) after addition of 40 μl luciferase assay reagent (Promega). β-Galactosidase activity was measured with a uQuant microplate reader (BioTek, Winooski, VT, USA) at 570 nm wavelength after addition of β-galactosidase assay reagent containing 1 mM chlorophenol red β-d-galactopyranoside substrate (Roche, Mannheim, Germany).

M. hyorhinis-specific serum IgG and IgM antibodies were evaluated every 7 days postinoculation using an in-house indirect enzyme-linked immunosorbent assay (ELISA). Briefly, M. hyorhinis cells were collected by centrifugation at 15,000 × g for 20 min at 4°C, washed, and resuspended in PBS. After lysis by ultrasonication, the supernatant was collected, and the protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime, China) according to the manufacturer’s instructions. The supernatant was diluted to 5 μg/mL with carbonate-bicarbonate buffer (pH 9.6) and used to coat 96-well ELISA plates overnight at 4°C. After blocking with 5% bovine serum albumin (BSA), each well was incubated with 100 μL of serum sample (1:100 in PBS containing 5% BSA) for 30 min at 37°C, followed by 100 μL horseradish peroxidase (HRP)-conjugated goat anti-swine IgG (Bethyl Laboratory, USA)/IgM (Bio-Rad, USA) (1:10,000 in PBS containing 5% BSA). After washing, the reaction was visualized by incubation with tetramethylbenzidine-hydrogen peroxide (TMB; Beyotime, China) substrate for 10 min at room temperature. The optical density (OD) of the solution was measured at 450 nm using a BioTek uQuant microplate reader (Bio-Tek, Winooski, VT, USA).

Enzyme-linked immunosorbent assays (ELISAs) were performed on stored plasma to determine the presence and levels of sCD14 (catalog number DC140; range, 250–16 000 pg/mL), IL-6 (catalog number S6050; range, 1.56–100 pg/mL), TNF-α (catalog number SSTA00C; range, 3.9–250 pg/mL), CRP (catalog number SCRP00; range, 0.78–50 ng/mL) (all from Quantikine immunoassays, R&D Systems Europe Ltd); IFABP (catalog number HK406-02; range, 47–3000 pg/mL), and EndoCAb (catalog number HK-504-IgM; range, 0.05–3.5 MMU/mL) (Hycult Biotech). Absorbance values were read using the BioTek U Quant microplate reader (catalog number 285179), and Gen5 software was used to interpret plate readings. Standard operating procedures were designed for each of these assays (according to the manufacturer’s recommendations) and validity of the results included assessing the R² of each standard curve (>0.9) and intra-assay/interassay validation %coefficient of variation of ≤ 15.

To determine the transcriptional activity of FOXO3, luciferase assay was performed as described previously,^{51 (link)} using FHRE-Luc, which was a gift from Michael Greenberg (Addgene plasmid # 1789).^{54 (link)} Briefly, using Lipofectamine 2000 (Invitrogen), cells were transfected with 100 ng of FHRE-Luc reporter, and 100 ng of indicated constructs, together with 20 ng of CMV/β-galactosidase plasmid to normalize transfection efficiency. After 24 h, cells were washed with PBS and permeabilized with Cell Culture Lysis Reagent (Promega). Luciferase activity was measured with a Turner Biosystems TD-20/20 luminometer after addition of 40 μl of luciferase assay reagent (Promega). β-galactosidase activity was measured with a uQuant microplate reader (BioTek, Winooski, VT, USA) at 570 nm wavelength after addition of β-galactosidase assay reagent containing 1 mM chlorophenol red β-d-galactopyranoside substrate (Roche).

Urease inhibitory potentials of the test samples were evaluated following previously reported procedure (Weatherburn, 1967 (link)). In brief, 25 μl enzyme solution and 55 μl of buffer (containing 100 mM urea) were incubated with 5 μl of test solution (1 mM concentration) in micro plate reader (room temperature for 15 min). Enzyme inhibitory potentials of our test samples were assessed from the rate of ammonia production. After 50 min of incubation, absorbance was recorded at 630 nm using Uquant Micro plate reader (BioTek Instruments Highland Park Winooski SN 213541 USA) using thiourea as control drug. Percent enzyme inhibition was calculated as;