M200 spectrophotometer

10-day-old imagos of both sexes from both strains were isolated on the day of the imaginal moult and placed in separate containers. After ten days, the hemolymph was collected. Five microliters of hemolymph were collected from the clipped leg of the 3rd pair and transferred to 10 µL of ice-cold PBS. A single sample contained hemolymph from three individuals (final volume 45 µL), and the number of replicates per group equalled 10. TRP and KYN content was measured using an ELISA assay performed using a commercially available kit (Kynurenine/Tryptophan ratio ELISA pack, ImmuSmol). The quantification was conducted with a Tecan M200 spectrophotometer.

The total phenolic content (TPC) of the obtained oils was determined using the Folin–Ciocalteu method [55 (link)]. The method was adapted for oils as follows: 2.5 g of oil was dissolved in 5 mL of hexane, and the phenolic compounds were extracted using 3 mL of MeOH:H₂O (60:40, v/v) for 1 min in a vortex system. Both phases were separated via centrifugation (at 3500 rpm for 10 min), and the lower phase was reextracted using 3 mL of MeOH:H₂O (60:40, v/v) following the same procedure. The methanolic extracts were pooled and an aliquot of 10 µL was taken to an Eppendorf vial and 0.6 mL of Milli-Q water and 50 µL of Folin–Ciocalteu reagent were added. After 3 min, 0.15 mL sodium carbonate solution (20%, w/v) was added to the reaction mixture, which was finally mixed and diluted with 1 mL of. The absorbance of the solution was measured after 2 h against a blank sample using a TECAN M200 spectrophotometer at a wavelength of 760 nm. The calibration curve was constructed using standard solutions of hydroxytyrosol (HT) within the range of 0–1 mg L⁻¹. These determinations were carried out in duplicate for each sample.

The total phenolic content (TPC) of the obtained oils was determined using the Folin–Ciocalteu method [55 (link)]. The method was adapted for oils as follows: 2.5 g of oil was dissolved in 5 mL of hexane, and the phenolic compounds were extracted using 3 mL of MeOH:H₂O (60:40, v/v) for 1 min in a vortex system. Both phases were separated via centrifugation (at 3500 rpm for 10 min), and the lower phase was reextracted using 3 mL of MeOH:H₂O (60:40, v/v) following the same procedure. The methanolic extracts were pooled and an aliquot of 10 µL was taken to an Eppendorf vial and 0.6 mL of Milli-Q water and 50 µL of Folin–Ciocalteu reagent were added. After 3 min, 0.15 mL sodium carbonate solution (20%, w/v) was added to the reaction mixture, which was finally mixed and diluted with 1 mL of. The absorbance of the solution was measured after 2 h against a blank sample using a TECAN M200 spectrophotometer at a wavelength of 760 nm. The calibration curve was constructed using standard solutions of hydroxytyrosol (HT) within the range of 0–1 mg L⁻¹. These determinations were carried out in duplicate for each sample.

The human chondrocyte cell line C28/I2 (gift from Mary Goldring, Cornell Medical College, New York, New York, USA; ref. 54 (link)) was utilized for micromass assays as described previously (20 (link)). Micromasses were stimulated with or without IL-1β (30 ng/ml) alone or in combination with 17R-RvD1 (0.1–100 nM) for 24 hours, and ECM accumulation was calculated following staining with Alcian blue as reported (21 (link)). Briefly, micromasses were fixed (4% glutaraldehyde, 15 min) acidified with HCl, and stained with Alcian blue 8GS overnight before dye extraction with guanidine hydrochloride (200 μl, 6M). Absorbance of extracted dye was measured at 620 nm using a Multiskan Bichromatic 348 spectrophotometer and normalized to DNA content using SYBRgreen dye (excitation 485 nm and emission 535 nm) with a TECAN M200 spectrophotometer. These values were used to generate ECM accumulation concentrations and are expressed as percentage change from control micromasses. In some experiments, the FPR2/ALX receptor antagonist WRW₄ (10 μM) was added to micromasses 10 minutes prior to 17R-RvD1 addition, and ECM accumulation was evaluated after 24 hours.

Filtration of 5 nm gold particles through nanocellulose filter papers was carried out in constant flow mode using an NE-1010 syringe pump (New Era Pump Systems, Farmingdale, NY, USA). Filtrations of gold particles were carried out at two different flux settings; 0.1 mL/min and 0.5 mL/min. The permeate solution was collected in fractions during the experiment and the real flux was monitored using a digital scale (Mettler Toledo, Columbus, OH, USA, MS1602TS). The collected fractions were analyzed using a TECAN M200 spectrophotometer. The absorbance was measured between 460–600 nm and the background absorption of the PBS solution was subtracted from the measurements. The area under the curve (AUC) was calculated for the absorption peak by integration over the measured wavelengths using MATLAB.
The particle removal efficiency is described by the logarithmic reduction value (LRV) and is calculated using Equation (1).
$\begin{array}{c}LRV=lo{g}_{10}\frac{AU{C}_{feed}}{AU{C}_{permeate}}\end{array}$
AUC_feed is the area under the curve for the feed solution and AUC_permeate is the area under the curve for the permeate solution. See Supporting Information for full procedure.