Spectramax plus microplate reader

The colon tissues were washed with pre-cooled physiological saline and dried with filter paper. Saline was then added at a ratio of 1:9. Next, the tissues were ground for 30 s and stopped for 30 s by a biological sample homogenizer (Aosheng Instrument, Hangzhou, China) three times, and then placed on ice for 5 min. After three cycles, the homogenate were centrifuged at 12,000 rpm/min at 4 °C for 20 min Finally, supernatants were collected to detect the expression of IL-1β, IL-6, TNF-α, IL-2, IL-10, IL-13, and sIgA by corresponding ELISA Kit (Multi Sciences, Hangzhou, China). In addition, the levels of serum CRP and D-lactic acid were determined by the CRP (Multi Sciences, Hangzhou, China) and D-lactic acid (Jiancheng Technology, Nanjing, China) kits according to the manufacturer’s instructions. The absorbance values were measured using Spectra Max Plus Microplate Reader (Molecular Devices, San Jose, USA) and the expression of each molecule was calculated according to the standard curve formula.

Overnight cultures of Salmonella in LB were diluted 1/1000 in LB or LB without NaCl supplemented with the appropriate antibiotics. Cells were grown in clear-bottomed 96-well black plates (Corning) using a SpectraMax Plus Microplate Reader (Molecular Devices) for agitation. Green fluorescence was measured using a Synergy H1 plate reader (BioTek) with 485-nm excitation and 535-nm emission, and the absorbance in each well was measured at 600 nm.

Serum anti-influenza levels were evaluated using peptides or live/inactivated influenza viruses, using the procedure previously described [14 (link)]. Briefly, 96-well NUNC MaxiSorp ELISA plates (Fisher Scientific, Pittsburg, PA, USA) were coated with peptides (1 µg/mL) or influenza viruses (10⁵ TCID₅₀ per mL) in PBS (100 µL per well) for 18–24 h at room temperature (RT) and 2–8 °C, respectively. Serial dilutions of serum samples starting at 1:100, with five-fold dilutions (1:100, 1:500, 1:2500, 1:12,500, etc.), were added to antigen-coated plates blocked with 3% normal goat serum (NGS, Southern Biotech, Birmingham, AL, USA) and detected with Horse Radish Peroxidase (HRP)-conjugated isotype-specific goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA). TMB Substrate Solution (Fisher Scientific, Pittsburg, PA, USA) was added prior to being quenched with TMB STOP solution (Fisher Scientific, Pittsburg, PA, USA). The absorbance values (450 nm) of each well were obtained using a SpectraMax Plus Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Data were expressed as endpoint titers defined as the reciprocal of highest dilution that yielded an absorbance value greater than or equal to twice that of the pooled pre-immune serum. Due to a minimum starting serum dilution of 1:100, titers below 100 were reported as nil.

The cytotoxicity of MeOH-E was tested in the normal NIH3T3 cells and HepG2 cells (1 × 10⁴ cells/well) cultured in DMEM composed of FBS (10%), antibiotic solution (1%) for 24 h at 37 °C in 5% of a CO₂ incubator. Later, the cells were treated with MeOH-E (0–100 µg/mL) for 24 h. After the treatment period, WST reagent (10 µL) was added, kept in a CO₂ incubator for 1 h, and then OD was measured at 450 nm using UV spectrophotometer (SpectraMax^® Plus Microplate Reader, Molecular Devices, San Jose, CA, USA). The percentage of cell toxicity was calculated by adopting the formula reported previously [78 (link)].

Aβ40 and Aβ42 levels were measured using an enzyme-linked immunosorbent assay (ELISA) (Thermo Scientific). The conditioned medium from cultured neurons was collected with 1× cOmplete protease inhibitor cocktail (Roche, Switzerland) and 1× PhosphoSTOP phosphatase inhibitor (Roche). Assays were performed following the manufacturer’s instructions. Absorbance at 450 nm was measured on a SpectraMax Plus Microplate Reader (Molecular Devices; San Jose, CA, USA). Each sample was measured in triplicate, and the final protein concentration was determined using a standard curve generated using the Aβ peptide standard provided in the assay kit.
The Aβ40 and Aβ42 enzyme-linked immunosorbent assays (ELISA) (Thermo Fischer Scientific) were performed as per the manufacturer’s instructions.