Hybrid microplate reader

We coated a 96-well plate (Nunc MaxiSorp, Nunc, Wiesbaden, Germany) with dilutions (1:500) of monoclonal anti-IFN-γ, anti-IL-12p40, or anti-IL-17A antibodies for 7 d at 4°C as previously described [20 (link)]. Next, the plates were blocked with 5% non-fat milk for 2 h at 25°C, the plates were incubated overnight at 4°C with the cell supernatant, or different dilutions of the recombinant IFN-γ, IL-17A, and IL-12p40. Following incubation with a biotinylated monoclonal IFN-γ, IL-17A, or IL-12p40 antibody, HRP-conjugated Streptavidin was added. The plates were washed three times with PBST at each step. Then, 3,3′,5,5′-tetramethylbenzidine (Thermo Scientific, USA) was used as the chemiluminescent substrate and the luminescence was measured using a Hybrid Microplate Reader (Epoch, BioTek Instruments, Inc, Winooski, VT, USA).

Mannan toxicity was assessed by reducing 3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) to a methanol-soluble formazan product. OREC were seeded in 96-well plates at a density of 2 × 10⁴ cells per well. OREC treatments were performed for 8 h using mannan at concentrations of 10, 50, 100, 200, 400 μg/mL. Additionally, OREC were stimulated with the optimal concentration of mannan (50 μg/mL) for 2, 4, 8, 12, and 24 h. Medium alone was used as the control. Then, the OREC were washed thrice with PBS and cultured in DMEM/F12 containing MTT (5 mg/mL) for 4 h at 37 °C. The medium was then discarded and the plates were gently shaken for 10 min in 100 μL of DMSO to extract the formazan. The optical density of the formazan was determined using the Hybrid microplate reader (BioTek Inc., Winooski, VT, USA) at 540 nm. The effect of different concentrations of mannan on OREC viability was determined by calculating the ratio between treated and untreated OREC.