Enspire multimode microplate reader

Supernatant from the cell culture of each experimental condition were collected for determination of the expression level of IL-6, TNF-α, and pro-inflammatory cytokines, by using a sandwich ELISA assay, following the manufacturer’s protocol (BioLegend, Inc., California, CA, USA). A 96-well plate was coated with capture antibody, specific to IL-6 and TNF-α at 4 °C for one night. The plate was washed four times with wash buffer between each step. The plate was then blocked with blocking solution for one hour with shaking. Supernatant from the cell culture of each condition and standard, were put into the reaction and shaken for two hours at room temperature. The plate was washed, followed by a specific binding of 100 µL of detection antibody specific to each cytokine, and incubated for one hour before washing. Avidin horseradish peroxidase-conjugated secondary antibody was then added and the plate was shaken for 30 min. The reaction was completed by adding 100 µL of freshly mixed solution of 3,3′,5,5′-Tetramethylbenzidine (TMB). After incubating for 15 min in the dark, stop solution was added. The absorbance of the reaction was measured at 450 nm using an EnSpire^® Multimode microplate reader (PerkinElmer, Inc., Massachusetts, MA, USA). The production of cytokines in each condition were calculated from standard curves using known concentrations of recombinant cytokines.

The sensitivity of the LAMP-CRISPR/Cas12a colorimetric assay was tested using 10-fold dilutions (1 ng–0.1 pg) of the verified DNA. Bovine, canine, chicken, duck, equine, ovine and porcine gDNA quality was verified using species-specific PCR primers (Table S1, Figure S4).
The assay specificity was determined using gDNA extracted from species or a mock preparation of fifteen mixed-meat products (Table S5) containing pork, chicken, duck, cattle, sheep, horse and dog meat. LAMP-CRISPR/Cas12a assays combined with these fifteen models with different mixing ratios were compared and analyzed with the naked eye under white light, blue light and ultraviolet light with immediate photography. The endpoint fluorescence signal intensity was analyzed using the EnSpire Multimode microplate reader (PerkinElmer, Akron, OH, USA).

A fluorometric Intracellular ROS kit (Deep Red) (Cat. No. MAK142; Sigma-Aldrich, Budapest, Hungary) was used to measure the oxidative stress in the cells. The experiments were carried out on a 96-well plate according to the manufacturer’s instructions. For the measurements, 5 × 10⁵ cells/well were treated with 10 ng/µL of 3′-epilutein or 3′-oxolutein and with 1 mM or 5 mM of glutamate or together 3′-epilutein or 3′-oxolutein first, followed by glutamate treatment. Briefly, the cells were rested for 24 h after seeding, then treated with 3′-epilutein or 3′-oxolutein or glutamate to generate ROS production in the cells. The treated plates were incubated for 10, 20, and 30 min in 5% CO₂ at 37 °C, then the cells were stained with 100 µL/well of ROS deep red dye solution and were incubated for 30 min. The absorbance was detected with a bottom read mode by EnSpire Multimode microplate reader (PerkinElmer, Rodgau, Germany) at 650/675 nm excitation/emission wavelengths. The alteration in ROS was determined as a percentage of control.

Measurement of oxidative stress was carried out with a Fluorometric Intracellular ROS Kit (Deep red) (Merck Life Science Kft., Budapest, Hungary). To generate ROS, 1 mM, and 5 mM of Glu were used in vitro in the SH-SY5Y cells. In the experiments, 5 × 10³ cells/well were plated onto a 96-well plate and rested for 24 h before treatments. Next, 10 ng/µL of lutein was used for the SH-SY5Y cells to examine the effect of lutein on ROS production alone and with the Glu treatments. In the mutual treatment, lutein was added first then immediately followed by the addition of Glu. The kit is able to detect the ROS in living cells. Each experiment has been completed according to the instruction of the manufacturer. Briefly, after the 24 h of resting, the cells were treated with 10 ng/µL of lutein and/or with the different concentrations of Glu to induce ROS production then the plates were incubated for 10, 20, and 30 min at 37 °C with 5% CO₂. Thereafter the cells were stained with 100 µL ROS deep red dye solution per well and were incubated for 30 min at 37 °C and 5% CO₂. EnSpire Multimode microplate reader (PerkinElmer, Rodgau, Germany) was used for the absorbance measurements with bottom read mode at 650 nm excitation and 675 nm emission wavelengths. The alteration in ROS was determined as a percentage of control.

Quantification of total MMP-9 and TIMP-1 was performed in KT patients at baseline, 7 days, 3 months, and 12 months post-KT using commercial ELISA Quantikine kits (R&D Systems, Minneapolis, MN, USA). MMP-9/TIMP-1 was calculated by dividing the levels of total MMP-9 by the levels of TIMP-1.
AlphaLISA^® technology was used to detect the interaction between MMP-9 and TIMP-1 following a published protocol [18 (link)]. Briefly, AlphaLISA^® acceptor beads (PerkinElmer, Waltham, MA, USA) were conjugated with an anti-MMP-9 antibody (ThermoFisher Scientific, Waltham, MA, USA) and then incubated with plasma samples from KT recipients and a biotinylated anti-TIMP-1 antibody (ThermoFisher Scientific). Streptavidin-coated donor beads were then added to bind the biotinylated anti-TIMP-1 antibody and detect the MMP-9/TIMP-1 interactions. Plates were read on an EnSpire Multimode Microplate Reader (PerkinElmer) using an excitation wavelength of 680 nm and an emission wavelength of 615 nm.

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging assay was used to evaluate the antioxidant activity of MO, CC extracts, MO lozenges and MO + CC lozenges. This assay was modified from a previous method [70 (link)]. A preparation of 0.1 mM DPPH solution was dissolved in 0.6 mM of stock solution with methanol. The extracts were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL and was added with 5 µL of extract in a 96 well plate. Then 50 µL of DPPH reagent (EMD Millipore Corp., Billerica, MA, USA) was added and the plate was incubated at room temperature for 15 min, after which the absorbance of the solution was measured at 540 nm using an EnSpire^® Multimode microplate reader (PerkinElmer, Inc., Waltham, MA, USA). A standard curve was obtained by Trolox (Sigma-Aldrich, St. Louis, MO, USA). The antioxidant activity of the MO extract was calculated as Trolox equivalent.