Spectramax i3

The total phenolics and flavonoid contents of freeze-dried radish microgreen samples were measured using the methodology implemented previously in our laboratory and described by Tilahun et al. (2022) (link). For total phenolics analysis, ethanolic extract (1 mg mL^-1) or standard was mixed with 1 mL of 10% Folin-Ciocalteu’s phenol reagent and 1 mL of 2% sodium carbonate solution. The absorbance at 750 nm was recorded using a microplate reader (Spectramax i3, Molecular Devices, Sunnyvale, CA, USA) after incubation of the samples at ambient temperature for 90 min in the dark. The results were expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample (mg GAE 100 g^-1) after comparison of the measurement to the calibration curve of gallic acid. For total flavonoids analysis, ethanolic extract (1 mg mL^-1) of the extract was mixed with 1.5 mL of ethanol, 0.1 mL of 10% aluminum nitrite solution, 0.1 mL of 1-M potassium acetate solution, and 2.8 mL of distilled water. The mixture was stirred and allowed to react for 30 min. Then, the absorbance was measured at 415 nm using a microplate reader (Spectramax i3, Molecular Devices, Sunnyvale, CA, USA). The measurements were compared to a quercetin (QE) calibration curve, and the results were expressed as milligrams of QE per 100 g of sample (mg QE 100 g^-1).

The total phenolics and flavonoids contents of freeze-dried peach fruit samples were measured in triplicate using the methodology implemented previously in our laboratory and described by Baek et al. (11 (link)). For the total phenolics analysis, ethanolic extract (1 mg ml^–1) or standard was mixed with 1 ml of 10% Folin–Ciocalteu’s phenol reagent and 1 ml of 2% sodium carbonate solution. The absorbance was measured at 750 nm using a microplate reader (Spectramax i3, Molecular Devices, Sunnyvale, CA, United States) after incubation of the samples at ambient temperature for 90 min in dark. A comparison of the measurement to the calibration curve of gallic acid was made, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of sample (mg GAE g^–1). For total flavonoids analysis, ethanolic extract (1 mg ml^–1) of the extract was mixed with 1.5 ml of ethanol, 0.1 ml of 10% aluminum nitrite solution, 0.1 ml of 1-M potassium acetate solution, and 2.8 ml distilled water. The mixture was stirred and allowed to react for 30 min. Then, the absorbance was measured at 415 nm using a microplate reader Spectramax i3, Molecular Devices, Sunnyvale, CA, United States). The measurements were compared to a quercetin (QE) calibration curve, and the results were expressed as milligrams of QE per gram of sample (mg QE g^–1).

Affinity sorbent (A19482, SPI Bio, Bertin Pharma) was used with a pool of different sources of plasma to prepare CGRP-free plasma. The samples were then analyzed using a commercially available CGRP kit (Catalogue No. A05482, SPI Bio, Bertin Pharma), following the manufacturer’s procedures. The concentration of CGRP was measured using Ellman’s Reagent to detect the enzymatic activity of the acetylcholinesterase, where the intensity of the yellow color formed was proportional to the amount of CGRP present in the sample (Castillo-Silva et al., 2019 (link)). The absorbance was read at 405 nm on SpectraMax I3 (Molecular Devices, San Jose, California, United States). The absorbance was read at 405 nm on a SpectraMax I3 (Molecular Devices, San Jose, California, United States). The absorbance values of the standards were used to plot a standard curve, from which the absorbance values of experimental samples were interpolated to determine their concentrations. This test was performed in mice plasma taken on the seventh-day post-stress induction.

The cell viability in 2D culture was assessed by Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) according to the manufacturer’s instructions. Briefly, SW480 cells were cultured with various concentrations of XAV939 for 72 h. Subsequently, 10 μL of CCK8 reagent was incubated with cells for 3 h, and then the absorbance at 450 nm was measured using a Spectramax® i3 (Molecular Devices). To determine the cell viability in 3D culture, a CytoSelect™ 96-Well Cell Transformation Assay (Cell Biolabs, Inc.) was performed according to the manufacturer’s instructions. Briefly, SW480 cells were embedded in 0.4% soft agar and treated with various concentrations of XAV939 for 6 days. The cells were then lysed and incubated with CyQuant GR dye. The fluorescence is proportional to the number of cells and was read using a Spectramax® i3 (Molecular Devices) with a 485/520-nm filter.

To evaluate cytotoxicity, 5 × 10⁴ luciferase engineered cells (NALM-6, SK-OV-3, LOX-IMVI, OVCAR-3) were co-cultured with T cells in a white flat bottom 96-well plate (Corning) at indicated effector:target for 6 or 24 h at 37 °C. After co-culture, 0.15 mg/mL D-Luciferin (Perkin Elmer, Waltham, MA) was added per well and luminescence was measured using a i3 SpectraMax (Molecular Devices, Sunnyvale, CA) across all wavelengths. Tumor cell viability was calculated as: ((Emission − Background)/(Tumor cell alone − Background)) × 100%. Each condition was tested in duplicate or triplicate.

Pre-adipocytes (3T3-L1, ATCC-173, Manassas, VA, USA) at a density of 1 × 10⁴/well were seeded in 96-well cell culture plates containing 10% FBS-DMEM 30-2002 medium (FBS-ATCC 30-2020 and DMEM ATCC 30-2002, Manassas, VA, USA) and incubated at 37 °C, 5% CO₂ for 24 h. After 24 h, the cells were treated with different concentrations of CMRO6 (0.98 µg/mL–500 µg/mL) for another 24 and 48 h under normal cell culture conditions. After that, 10µL of EZ-cytox reagent (DoGenBio, Seoul, South Korea # EZ-1000) was added to each well and incubated for 30 min to 60 min, and optical intensity was measured at a 450 nm (i3 Spectramax (Molecular Device, San Jose, CA, USA [15 (link)].