Quant microplate spectrophotometer

The total phenolic content (TPC) was determined using the modified version of the Folin-Ciocalteu method previously reported [69 (link)]. Briefly, 50 μL of Folin-Ciocalteu (50%, v/v) reagent and 400 μL of Na₂CO₃ (5%, w/v) were added to 50 μL of the test sample, in a 48-well plate, in triplicate. The plate was shaken, incubated at room temperature for 20 min (in the dark) and the absorbance was measured at 760 nm (in a µQuant™ Microplate spectrophotometer, BioTek Instruments Inc., Bad Friedrichshall, Germany), against a blank consisting of a mixture of the reagent’s solvents. In addition, gallic acid (GA) was used as a reference standard (in the 5–60 μg/mL concentration range) and the TPC was expressed as mean ± standard deviation of three independent experiments as equivalents of gallic acid per gram of extract (mg GAE/g extract).
The above-described procedure was also applied to the BSO extract, following the methodology reported by other authors [70 (link)] which enables to directly quantify the oil sample after solubilization in an appropriate solvent, without previous extraction of the phenolic compounds. The currently prepared ethanolic solution of the seed oil was easily mixed with the aqueous Folin-Ciocalteau reagent, with no precipitates having been formed, and thus allowing accurate spectrophotometric readings.

Elemental analysis was carried out at the Chemistry Department of the University of Aveiro (by M. Marques) on a Leco CHNS TruSpec^® Micro elemental analyzer. Laboratory powder XRD data were collected at ambient temperature on an X’Pert MPD Philips diffractometer (Cu Kα₁ = 1.540598 Å) with a curved graphite monochromator, equipped with a X’Celerator detector, operating in a flat Bragg–Brentano configuration (40 kV, 50 mA). Data was collected with steps of 0.04° in a continuous mode in the 3.5° ≤ 2θ ≤ 50° interval. TG studies were performed on a Shimadzu TGA-50 thermogravimetric analyzer, using a heating rate of 5 °C min⁻¹, under air atmosphere, with a flow rate of 20 mL min⁻¹. The sample holder was a 5 mm in diameter platinum plate and the sample mass was about 5 mg. Infrared spectra were obtained as KBr pellets in a Mattson 7000 FTIR spectrometer (resolution 2.0 cm⁻¹; 64 scans per spectrum). ¹³C{¹H} CP/MAS NMR spectra were recorded at 100.62 MHz on a (9.4 T) Bruker Avance III 400 spectrometer, with an optimized π/2 pulse for ¹H of 4.5 μs, 3 ms contact time, a spinning rate of 12 kHz and 4 s recycle delays. Chemical shifts are quoted in parts per million from tetramethylsilane. Absorbance readings for the antioxidant activity assay were measured at the working wavelength of 515 nm in a µQuant™ Microplate spectrophotometer by BioTek Instruments, Inc.

D-Dimer in plasma (blood was collected via retro-orbital bleed in 3.8% sodium citrate) was quantified using Asserachrom D-Di enzyme immunoassay kit (Diagnostica Stago, Asnières, France) following the manufacturer's protocol. Samples were read at 450 nm using a µQuant microplate spectrophotometer (Bio-Tek Instruments, Winooski, VT).

CT26 and KHT cells were seeded at a density of 1000 and 1500 cells/well, respectively. The following day, cells were treated with varying concentrations of IFNα (PeproTech, UK). Media was replaced after 24 or 48 hr, or was not removed until the experimental endpoint at 72 hr. After 72 hr, 50 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) was added to wells for 4 hr, after which the media were replaced with DMSO for analysis of optical density using a µQuant Microplate Spectrophotometer (BioTek, UK) at a wavelength of 540 nm.

Aqueous ethanolic extracts from each tamarillo genotype were used for the antioxidant analyses. For each assay, three independent experiments were performed. The samples were measured in a multi-well spectrophotometer (µQuant™ Microplate spectrophotometer, BioTek Instruments Inc., Bad Friedrichshall, Germany). The blanks consisted of a mixture of the reagent’s solvents. The protocols followed to assess the antioxidant capacity were the same as described in Marques et al. (2021) [17 (link)].