Spectra id3 max

Mouse melanoma B16F10 cells (American Type Culture Collection (ATCC, CRL-6475, Manassas, VA, USA) were cultured under standard conditions (37 °C, 5% CO₂, 95% humidity) in Ham’s F-10 Nutrient Mix (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and antibiotics (1% streptomycin/penicillin mixture; Sigma–Aldrich, Darmstadt, Germany). The cells were seeded at the density of 2 × 10³ cells/mL on 96-well plates in Ham’s F-10 Nutrient Mix, 1% FBS, and 1% antibiotics. After 24 h, the cells were treated with the tested extracts or kojic acid and incubated for an additional 48 h. Next, MTT reagent (Sigma–Aldrich, Darmstadt, Germany) was added to each well and further incubated for 4 h. The formazan produced in the cells appeared as dark crystals at the bottom of the wells, which could be observed using an inverted microscope. Next, the medium was aspirated, and 100 µL of DMSO was added to each well. The absorbance was measured at 570 nm (A570) using a plate reader (Spectra iD3 Max, Molecular Devices; San Jose, CA, USA). Viability (% of control) was determined by dividing A570 of experimental wells (after incubation with the tested extracts) × 100% by A570 of control wells (after incubation with the solvent). Experiments were performed in triplicates [51 (link),52 (link)].

To investigate the preliminary hepatic safety of HBK-10 at the concentration used in metabolic stability study, a human hepatocellular carcinoma cell line—HepG2 (HB-8065TM, ATCC, Manassas, VA, USA) was used in the study. HepG2 cells were cultured in standard conditions (37 °C, 5% CO₂, 95% humidity) in appropriate culture medium (according to manufacturer procedure), supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (1% streptomycin/penicillin mixture; Sigma-Aldrich, Steinheim, Germany). Cells were seeded in 96-well plates at density of 1 × 10⁴ per well. After 24 h cells were treated with two doses of HBK-10 (5 and 25 µM, solvent—PBS) and incubated for additional 24 h. Following the incubation cells viability was measured with MTT assay. For this purpose, MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma Aldrich, Steinheim, Germany) was added to each well at the concentration 0.5 mg/mL. After 4 h incubation at 37 °C, the medium was aspirated, and formazan crystals produced in the cells were dissolved in DMSO. Then the absorbance of solution was determined at 570 nm (A570) on plate reader (Spectra iD3 Max, Molecular Devices; San Jose, CA, USA). Viability (% of control) was determined by dividing A570 of experimental wells by of A570 of control wells × 100%.

Mouse melanoma B16F10 cells (American Type Culture Collection (ATCC, CRL-6475, Manassas, VA, USA) were seeded at the density of 5 × 10⁴ cells/mL on 24-well plates and incubated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 1% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) and antibiotics (1% streptomycin/penicillin mixture; Sigma–Aldrich, Darmstadt, Germany). After 24 h, the medium was replaced with new samples, containing either the tested extracts, a reference inhibitor, or a solvent with or without the addition of 1 µM α-MSH (Sigma–Aldrich, Darmstadt, Germany). The cells were further incubated under these conditions for 48 h. The cells were then washed twice with PBS and dissolved in 100 µL of 1M NaOH. Next, the plates were incubated at 60 °C for 1 h and mixed to solubilize melanin. The samples were then transferred to a 96-well plate, and the absorbance value was measured at 405 nm using a plate reader (Spectra iD3 Max, Molecular Devices, San Jose, CA, USA). The relative melanin content ± SD was found for 100%, and assigned to cells treated neither with extract nor kojic acid, with the absence of α-MSH [51 (link)].