Multiskan skyhigh microplate spectrophotometer

To evaluate the effect of the Tri-TT on milk production, AQP3, AQP5, and LALBA levels in plasma were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Sunlong Biotech Co., Ltd., Hangzhou, China). AQP3, AQP5, and LALBA levels were examined using the microelisa strip plate that was pre-coated with an antibody specific to AQP3 or AQP5, or LALBA. Standards or samples were added into wells and combined with the specific antibody. Horseradish peroxidase (HRP) conjugated antibody-specific protein was added to each well and incubated. After washing, TMB substrate solution was added into the wells to detect the reaction, the color changed to yellow. After the addition of the stop solution, the optical density (OD) was measured spectrophotometrically at an absorbance wavelength of 450 nm by a microplate reader (Thermo Scientific™Multiskan SkyHigh Microplate Spectrophotometer). The mean OD values were derived from the three replicates of the ELISA analysis. The sensitivity of AQP3, AQP5, and LALBA ELISA kits was 6 pg/ml, 25 pg/ml, and 0.01 ng/ml respectively.

Cell viability was measured using Cell Proliferation Reagent WST-1 (Roche, Basel, BS, Switzerland). In brief, Meg-01 cells were seeded at a density of 1000 cells per well in 100 μL culture medium containing gradient concentrations of A. cinnamomea extracts in 96-well plates. Cells cultured in a medium without A. cinnamomea extract were used as control cells. The plates were incubated at 37 °C in an incubator with 5% CO₂ for 7 days. After incubation, 10 μL of WST-1 assay reagent was added to each well and incubated for another 2 h. Absorbance at 440 nm was determined using theenzyme-linked immunosorbent assay (ELISA) reader (Multiskan SkyHigh Microplate Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). All experiments were performed in duplicate three times. According previous studies described [64 (link),65 (link),66 (link)], the values of CC₁₀ and CC₅₀ (cytotoxicity concentration that causes a 10% and 50% reduction in cell numbers compared to the control) were calculated a 4-parameter logistic (4PL) model and were regarded as the working concentration of the A. cinnamomea extracts for the virus inhibition assay.

The R. ferrugineus were homogenized larvae in a buffer (sodium phosphate, 40 mM, and pH 7.4); then, sodium chloride (10 mM dissolved in 1% (w/v) triton X-100) was added. During centrifugation (Sigma 3–30 KS), the homogenate was chilled to 4 °C and spun at a speed of 5000 rpm (20 min). The supernatant was either immediately put into action for an enzymatic test or frozen and kept at 20 °C for later use. Each test was repeated three times. The protein content was determined using the same protocol employed by Lowry with modification [93 (link)] using protein extract (100 µL) combined with an alkaline copper solution and a Folin–Ciocalteu phenol reagent (2 mL and 200 µL, respectively). The intensity of the resulting blue hue was evaluated during 30 min of incubation at 25 °C. Using an ELISA plate reader and a BSA standard curve, we could calculate the protein concentration by analyzing the absorbance at 600 nm (Multiskan SkyHigh Microplate Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA).

The method proposed by Guo et al. [11 (link)] based on the Liebermann–Burchard reaction [12 (link)] allowed the determination of the type of saponins present in the sample. For steroidal saponins, the reaction becomes yellow–green, while for triterpenic saponins, the reaction will develop a pink–red coloration. For this, a small amount of extract was mixed with acetic anhydrous:chloroform in a 1:1 ratio. Then, three drops of concentrated sulfuric acid (72% wt.) with vanillin (0.5% wt.) were added, they were left to react for 10 min at 60 °C, followed by a cold-water bath for 15 min, and later colorimetric observations were made using an ultraviolet–visible (UV-VIS) spectrophotometer Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Total RNA was isolated from PBMCs using the Direct-zol™ RNA Miniprep (Zymo Research., CA, USA), then quantified using a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Hanover, MD, USA), and cDNA synthesis was done with 150 ng of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems^™, CA, USA) following the manufacturers’ recommendations. The expression of IL-1β, IL-6, IL-8, and TNFα, as well as of the phosphoglycerate kinase (PGK) used as reference gene, was quantified by real-time RT-PCR using the Maxima SYBR^® Green qPCR master mix (Thermo Fisher Scientific, Hanover, MD, USA), and a CFX-96 Real-Time thermal cycler (Bio-Rad, Hercules, CA, USA) as previously described ¹⁹ (link). Oligos and thermal conditions are shown in supplementary material 1. Real-time RT-PCR analysis was conducted using the CFX Maestro 1.1 Software (Bio-Rad).

The cytotoxicity of terfenadine was evaluated on the Caco-2 cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, based on a reduction in MTT to purple formazan granules. The MTT assay was performed as described previously by Mossman [51 (link)], with slight adjustments. Briefly, Caco-2 cells were seeded into a 96-well plate (5000 cells/well) and grown at 37 °C in 5% CO₂ for 24 h. Cells were then treated with DMSO (0.09%, the negative control) or terfenadine (1, 2, 3, 16, and 32 μM). After 24 h, the medium was removed, 100 µL of MTT reagent (0.8 mg/mL in serum-free medium) was added to each well, and the cells were incubated at 37 °C for 4 h in 5% CO₂–95% air atmosphere. Next, the medium was replaced with DMSO (150 µL) to dissolve the formazan crystals, and the absorbance was measured at 570 nm on a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer, A51119500C, Thermo Scientific, Waltham, MA, USA).