Magellan software

The total phenolic content (TPC) of the various solvent extracts of T. ferdinandiana tissues was determined by spectrometry using the Folin–Ciocalteu reagent [19 (link)]. The extracts (25 µL) were added to the 96-well plate and 125 µL freshly prepared Folin–Ciocalteu reagent and 125 µL sodium carbonate (7.5% w/v) were also added to the wells. The mixture was incubated in the dark for 30 min at room temperature. Absorbance was measured at 750 nm using a Tecan Microplate Reader (Tecan Infinite M200, Tecan Trading AG, Mannedorf, Switzerland) with Magellan Software (version 6.4, Tecan Trading AG). Results were expressed as g gallic acid equivalents (GAE)/100 g dry weight (DW).

Samples were tested by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Assays were performed with the commercially available equipment and products of BioReba (Reinach, Switzerland) following the protocols provided by the manufacturer. One gram of pooled wood samples was arranged in extraction bags and homogenized in 1:10 (w/v) customized “Grapevine” extraction buffer using the homogenizer HOMEX (BioReba, Reinach, Switzerland). Scion samples were tested for ArMV, GFLV, GLRaV-1, and GLRaV-3 following the manufacturer’s recommendations. Samples from rootstock plants were also tested for GFkV. In 2018, scion and rootstock samples were additionally tested for GPGV. Positive and negative controls were obtained from the WBI virus collection located in Freiburg, Germany. ELISA plates were evaluated photometrically after 30 and 60 min, using an Infinite F50 reader and Magellan™ software (Tecan Trading AG, Maennedorf, Switzerland). Samples were considered positive if the absorbance value was twice the value of the negative control sample.

Serum concentration of RANKL was measured by commercial ELISA kits (DY626, R&D Systems, Minneapolis, MN, USA) according to protocols provided by the manufacturer. The analyses were performed for a subgroup of RA patients consisting of 109 individuals before anti-TNF treatment and 99 controls. The absorbance was measured in a Tecan Sunrise absorbance reader and Magellan software (Tecan Trading AG, Switzerland). The optical density of each well run in duplicate was determined by microplate reader set to 450 nm with wavelength correction set to 570 nm. Peptide concentration in the samples was measured by comparing the optical density of the sample with a computer-generated four parameters logistic curve-fit standard curve.

Matrix MetalloProteinase 13 (MMP13), a Disintegrin And Metalloproteinase with Thrombospondin motif 5 or Aggrecanase-2 (ADAMTS-5), Aggrecan (Aggr), Cartilage Oligomeric Matrix Protein (COMP), Procollagen II C-Terminal Propeptide (PIICP), Chondroitin Sulfate (CS), and Collagen Type 2 Cleavage (COL-2CAV) were measured by ELISA in culture supernatants recovered after compression, according to manufacturer’s instructions (Cloud-Clone Corp, United States for MMP13, ADAMTS-5, Aggr, COMP, PIICP and CS kits; MyBioSource, United States for COL-2CAV) using Infinite^® M Plex multimode microplate reader equipped with Magellan Software (Tecan Trading AG, Switzerland) and a programmable microplate washer (Hydro flex Tecan Trading AG, Switzerland). Results were normalized to mg of tissue of the corresponding explant. Due to bioreactor technical requirements, each cartilage cylinder was cultured in 2 ml medium, resulting in high dilution of released factors in supernatants. For this reason, supernatants were vacuum concentrated before ELISA test.

Rat soluble fms-like tyrosine kinase-1 (sFlt-1, #MBS2602003) and placenta growth factor (PLFG, #MBS026910) were tested in placenta and serum with enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instruction (Mybiosource). In brief, 1-mg protein of rat placenta (homogenized in Pierce™ IP lysis buffer containing 1% (v/v) protease inhibitor cocktail) and 100-μl serum were added as samples. Samples and standard were then covered and incubated at room temperature for 90 min with gentle shaking. After washing twice, biotinylated antibodies were added and incubated at room temperature for another 60 min. After washing for three times, HRP-Avidin was added and incubated for 30 min at room temperature with gentle shaking. After washing for five times, color reagent was added in dark with gentle shaking. After 30 min of incubation, stop solution was added and the absorption was read at 450 nm immediately with Sunrise™ microplate reader (Tecan Group) and analyzed by Magellan™ software (Tecan Group).

Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) assay or trypan blue exclusion assay. MTT assay was performed as previously described^{56 (link)}. Briefly, cells (2.5 × 10³ cells/well) were seeded in each well of a 96-well plate and incubated for 24 h. The cells were cultured in amino acid-depletion media for an additional 72 h. Thereafter, 50 μL of MTT (2 mg/mL in PBS) was added to each well and the plates were incubated for an additional 2 h. The resulting formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO) after aspiration of the culture medium. Plates were placed on a plate shaker for 1 min and read immediately at 570 nm using a TECAN micro-plate reader with Magellan software (Tecan Group Ltd., Männedorf, Switzerland).
The trypan blue exclusion assay was performed by seeding 1 × 10⁵ cells/well into 12-well cell culture plates and incubating them for 24 h at 37 °C. Seventy-two hours after P4HA3 siRNA transfection, cells were disaggregated in 500 μL medium and 10 μL of the suspension was mixed with 10 μL trypan blue (Thermo Fisher Scientific). Viable cell counts were obtained using the Countess Automated Cell Counter (Thermo Fisher Scientific).