Sunrise elisa plate reader

Tissue were homogenized and diluted in saline at a final concentration of 50 mg/mL as previously described^{61 (link), 65 (link)}. Homogenates were diluted (1:1) in 1% deoxycholate (Sigma-Aldrich) and incubated at 37 °C for 5 min. For triglyceride measurements, samples were diluted 1:100 in the reagent (Infinity™ Triglycerides Liquid Stable Reagent, Thermo Fisher Scientific) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm with a Sunrise ELISA plate reader (Tecan, Männedorf, Switzerland). Concentrations were determined compared with a standard curve of triglycerides (Infinity™ Triglycerides Standard, Thermo Fisher Scientific). The protein content of the preparations was measured by the Bradford method, using BSA (Sigma-Aldrich) as standard. All assays were performed in duplicate.

The hepatic triglyceride content was measured by enzymatic methods, in accordance with previously published procedures [22] (link). Briefly, tissues were homogenized and diluted in saline at a final concentration of 50 mg/mL. Homogenates were diluted (1∶1) in 1% deoxycholate (Sigma) and incubated at 37 °C for 5 min. For triglyceride measurements, samples were diluted 1∶100 in the reagent (Infinity Triglycerides Liquid Stable Reagent, Thermo Electron) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm with a Sunrise ELISA plate reader (Tecan). Concentrations were determined compared with a standard curve of triglycerides (Infinity Triglycerides Standard, Thermo Electron). The protein content of the preparations was measured by the Bradford method, using BSA (Sigma) as standard. All assays were performed in duplicate.

Total phenol content of the yerba mate tea was estimated by a colorimetric assay based on the procedure described by Kumazawa et al. [23 (link)]. The 0.1 mL of yerba mate tea solution (100~500 ppm) was mixed with 0.5 mL of Folin-Ciocalteu reagent for a duration of 1 minute and 0.5 mL of 2% sodium carbonate for 20 minutes. The absorbance at 655 nm (Sunrise ELISA plate reader, Tecan, Austria) increases with the total phenol content.

At the end of the experiment, glycogen concentration was measured using a Glycogen Assay Kit (MAK016, Sigma, Germany), according to the manufacturer's protocol. Samples were measured using the Sunrise ELISA plate reader (Tecan, Germany)

For analysis of Gremlin-1 plasma levels, peripheral blood samples of LDS patients and healthy donors were collected and centrifuged for 10 min at 2,000 g in order to separate the plasma from the blood cells. Gremlin-1 protein levels were measured using the enzyme-linked immunosorbent assay (ELISA) Kit for Gremlin-1 (Uscn Life Science Inc., Missouri City, TX) following the instructor's manual. Absorbance of color change was quantified with the Sunrise ELISA plate reader and Magellan software (Tecan, Maennedorf, Switzerland).

RHDV-specific antibodies in immunized rabbits were detected by indirect ELISA assays. To prepare the encapsulated antigen, PK15 cells were infected with rSWPV-VP60-His, maintained in serum-free DMEM for 5 days, and the freeze–thaw supernatant was collected to purify the VP60-His protein according to the steps of the His-tagged protein purification kit (CWBIO, China). The purified recombinant VP60-his protein was used to coat 96-well plates at a concentration of 5 μg/ml in a volume of 100 μl per well. Test sera were titrated in duplicate using 1,000-fold dilutions. The plates were incubated at room temperature for 1 h, then washed and incubated for 1 h with detection antibody, goat anti-mouse IgG-horseradish peroxidase (HRP; 1:2000 dilution; TransGen, China). The plates were washed and incubated using 3,3′,5,5′-tetramethylbenzidine (TMB) at room temperature in the dark; the reaction was stopped after 15 min using 1 M H₂SO₄ and read out at 450 nm (Tecan Sunrise ELISA plate reader). The sera from the rabbit inoculated with inactivated SWPV-JX20G were used as the negative control the results are expressed as OD₄₅₀ nm values.

Briefly, 10 μL of AuNP solution, 45 μL of ultrapure water, and 0.5 μL of I⁻ solution (4 mM) were mixed. After allowing them to react for 15 min, 40 μL of different concentrations of cysteine solution were added to the mixture. After 15 min, 2 μL of H₂O₂ (10 M) and 2.5 μL of TMB solution (6 mM) were added into the reaction solution. After 5 min, 5 μL of stop solution was added to the solution. The absorption spectra were measured at 450 nm after 2.5 min using a Sunrise ELISA plate reader (Tecan Austria GmbH, Salzburg, Austria).
To understand the catalytic state of AuNPs, the normalized absorbance was calculated as ΔA450/A450₀ (ΔA450  = A450₀ − A450_x), where A450₀ and A450_x were the absorbance intensities of the AuNP system before and after the addition of cysteine, respectively. The error bars were obtained by calculating the standard deviation of the normalized absorbance values and from three independent experiments in the same 96-well microplate.