Infinite 200 pro series

The DPPH radical scavenging assay (RSC) was performed according to a previously described method [42 (link)]. Briefly, 1 mL of freshly prepared ethanolic solution of DPPH radical (100 μM) was mixed with the tested extract solution in EtOH at various concentrations (0.5, 1.0, 2.0, and 4.0 mg/mL). The mixture was then vortexed and incubated at room temperature in the dark for 30 min, followed by absorbance measurement at 517 nm on the reader Infinite^® 200 PRO series (Tecan Group Ltd., Männedorf, Switzerland). Experiments were performed in triplicate for each sample and twice in total. Gallic acid was used as a positive control at a concentration of 30 μM. A negative control with 10 μL DMSO and 190 μL DPPH was performed each time. The blank solution contained 190 μL EtOH and 10 μL sample. The radical scavenging activity percentage (AA%) was calculated as follows: AA% = [1 − ((A_sample − A_blank)/A_control)] × 100, where A_control is the absorbance of the negative control, A_sample is the absorbance after the reaction of samples with DPPH, and A_blank is the absorbance of sample with EtOH instead of DPPH. Moreover, the IC₅₀ value indicating the extract amount that caused 50% scavenging of the DPPH radical was calculated.

The external glucose and glycerol concentrations were measured using the commercially available D‐glucose HK and Glycerol Assay Kits, respectively (Megazyme, Ireland). Sampling was performed during the microplate growth experiments described above. An appropriate well (150 μl) was sacrificed at regular time intervals, the culture volume centrifuged at 14,000 g, and the supernatant stored at −20°C. The glucose and glycerol concentrations were determined from absorbance measurements at 340 nm in a microplate reader (Infinite 200 PRO series, Tecan Group Ltd., Germany), by means of a calibration curve and appropriate dilutions, following the manufacturer's instructions.

Proliferating normal human diploid fibroblasts (BJ cells) were cultured as described before [35 (link)]. In order to evaluate elastase activity, the amount of released p-nitroaniline, which was hydrolyzed from the substrate (N-succinyl-Ala-Ala-Ala-p-nitroanilide) by elastase, was determined by measuring the absorbance at 405 nm. Thus, BJ fibroblasts were seeded into 96-well microplates and the next day they were treated with compounds 1 to 7 at a final concentration of 5 μM for each compound and of 1 μg/mL or 10 μg/mL of concentration for each extract, for 24 h. Afterwards, cells were lysed in 100 mM Tris-HCl (pH 7.6) with 0.1% Triton X-100 buffer and subsequently, 2 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma-Aldrich) was added to each well followed by incubation at 37 °C for 1 h. The absorbance at 405 nm was measured using a microplate reader Infinite 200 PRO series (Tecan Trading AG, Switzerland).

HDFs were seeded in 96 well plates—5000 cells/well—in 100 µL of complete medium. After 8 h of incubation, cells were washed with PBS and then cultured overnight with serum-free medium. After that, cells were washed with PBS and EVs were administered—1.5 × 10¹¹ EVs/mL of P10K and 2 × 10¹¹ and 4 × 10¹¹ EVs/mL of P100K—in 100 µL of 1:5 complete medium in serum-free medium and incubated for 24 h. Complete medium was used as positive control. After that, cells were washed with PBS and 100 µL of CCK8 (Merck, Cat: 96992) 1:10 diluted in culture medium were added. After 4 h of incubation, absorbance was read with a plate reader (Infinite® 200 PRO series, TecanTrading AG, Männedorf, Switzerland) at 450 nm, using 650 nm as the reference wavelength.

Cytotoxicity assays were performed following the ISO 10993-5:2009 guidelines for biological evaluation of medical devices.
HaCaT and HDFa (5000 cells/well) and M0 macrophages (20,000 cells/well) were seeded into a 96-well plate on modified culture media supplemented with FBS. All plates were incubated for 1 h in 5% CO₂ at 37 °C to allow complete cell attachment and stretching. Next, different NCM concentrations (0.1–200 NCM/cell) were added and incubated for 48 h. Dimethyl sulfoxide (DMSO, 10%), was used as cytotoxicity control. After 48 h, NCMs were removed from cultures and CCK-8 reagent (Sigma-Aldrich, Saint Louise, MO, USA) added and incubated for 4 h. Absorbance was read with a plate reader (Infinite^® 200 PRO series, Tecan Trading AG, Männedorf, Switzerland) at 450 nm and at 650 nm, as the reference wavelength. Based on these results, the NCM/cell ratios were established for further experiments.

The anti-tyrosinase capacity of the extracts or compounds was assayed using an enzymatic method as previously described [45 (link)] with minor modifications. Briefly, in a 96-well microplate, 1/15 M PBS (pH 6.8), the tested samples, and 92 U/mL of mushroom tyrosinase (Sigma-Aldrich) were mixed and incubated for 10 min at room temperature, avoiding light exposure. Following the addition of 2.5 mM L-DOPA (Sigma-Aldrich), the mixture was incubated at 25 °C for 5 min. The absorbance at 475 nm of each well was measured using reader Infinite 200 PRO series (Tecan Trading AG, Switzerland) and the MagellanTM software. The percentage inhibition of the tyrosinase activity was calculated by the following equation: [(A − B) − (C − D)]/(A − B) × 100, where A is the control (w/o sample), B is the blank (w/o sample, w/o tyrosinase), C is the sample, and D is the blank sample (w/o tyrosinase). Blanks contained all the aforementioned components except for the enzyme. Kojic acid (KA) and the methanolic extract from the root of Glycyrrhiza glabra L. (Gly) were used as positive controls at final concentrations of 2 and 5 µg/mL, respectively.