Microplate spectrophotometer

Each 0.2 g sample was flash-frozen in liquid nitrogen, pulverized, combined with 10 mL of 80% (v/v) methanol, vortexed for 15 s, sonicated for 20 min, and centrifuged at 3000× g and 22 °C for 20 min. Each supernatant was transferred to a new 15 mL tube, diluted with an equal volume of 80% (v/v) methanol, and used in the subsequent assays.
Total phenolic content was determined with Folin–Ciocalteu reagent [52 ]. Fifty microliters diluted solution and 50 μL Folin–Ciocalteu reagent were added to 450 μL distilled water. The mixture was vortexed briefly and incubated at 22 °C for 5 min. Then, 150 μL of 20% (w/v) Na₂CO₃ and 200 μL distilled water were added, and the mixture was incubated in the dark at 22 °C for 30 min. Absorbance was measured at 750 nm using a microplate spectrophotometer (BioTek Epoch, Winooski, VT, USA). A standard curve was plotted using various gallic acid dilutions.
Total antioxidant activity was determined by evaluating the ABTS^•+ scavenging activity [53 (link)]. The ABTS^•+ solution was diluted with distilled water to OD₇₃₄ = 0.7. Then, 1.2 mL diluted ABTS^•+ solution was mixed with 10 μL sample. The mixture was vortexed briefly and incubated in the dark at 22 °C for 15 min. Absorbance was measured at 734 nm using a microplate spectrophotometer (BioTek Epoch, Winooski, VT, USA). A standard curve was constructed using various Trolox dilutions.

The cellular tyrosinase activity was conducted as previously described (Lv et al., 2020 (link)). SK-MEL-2 cells were incubated with different concentrations of FGIN-1-27 (0, 1, 2, 4 μM) or OAG (200 μM) for 12 h or 48 h and then lysed with cell lysis buffer. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C to obtain the supernatant for tyrosinase activity assay. Protein concentrations were determined by BCA kit with bovine serum albumin (BSA) as a standard.100 μL PBS (0.1 M, pH 6.5) containing 30 μg proteins mixed with 100 μL L-DOPA (0.1%) and then incubated at 37°C for 1 h. Optical absorbance was measured at 475 nm using a microplate spectrophotometer (BioTek Instruments).
The direct effect of FGIN-1-27 on tyrosinase activity was conducted in accordance with a previous method (Tomita et al., 2010 (link); Lv et al., 2020 (link)). 100 μL PBS (0.1 M, pH 6.5) containing different concentrations of FGIN-1-27 mixed with mushroom tyrosinase (10 unit) and 50 μL L-tyrosine (0.05%) and then incubated at 37°C for 10 min. Optical absorbance was measured at 475 nm using a microplate spectrophotometer (BioTek Instruments).

Cells were dissociated with Accutase and plated at 10⁴ cells/well in 96 well plates. After overnight culture, graded doses from 0 to 3.3 plaque-forming units (PFU) per cell of G207 or M002 were added to each row and cytotoxicity was measured 72 hours post-infection with alamarBlue® (Life Technologies, Grand Island, NY) as previously described^{23 (link),25 (link),50 (link)}. Graded doses of the virus were internally compared to mock-treated controls, which were included with each experiment and represented 100% tumor cell survival. Color changes of alamarBlue® were quantified with a BioTek microplate spectrophotometer (Winooski, VT), and OD595-562 nm values were used to calculate the IC₅₀. (N ≥ 4).

The specimens with the treated biofilms (5 per group) were transferred to Falcon tubes containing 5 mL of PBS and incubated at 37 °C for 3 h. Then, 1 mL of each tube was placed in a freezer at −80 °C for 5 min to stop the acid production. To verify the concentrations of lactate, the enzymatic method of lactic dehydrogenase was used according to the manufacturer’s instructions (Lactic dehydrogenase LDH UV K014, Bioclin, Belo Horizonte, Brazil). Absorbance was measured at 340 nm using a microplate spectrophotometer (Epoch, Biotek, Winooski, VT, USA), and the obtained values were expressed as mmol lactate/L.