Spectramax m

To assess the effect of PTH1-34, ATDC5 cells were cultured in 96-well multiplates at 5 × 10³cells per well, followed by incubation with different concentrations of PTH1-34 or the same volume of NS for 6 h per 24 h after cell attachment. Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was applied to measure the absorbance at day 1–6 timepoints using SpectraMaxM (Molecular Devices, San Jose, CA, United States), according to the manufacturer’s instructions.

The right lungs (from the same animals used for histological study) were macerated for nitric oxide (NO) and cytokine measurements in lung tissue homogenates. NO production was evaluated according to Griess method (Green et al., 1982 (link)), and fluorescence was measured at 570 nm wavelength (SpectraMax M, Molecular Devices, San Jose, CA, United States).
Cytokine concentrations (IL-1β, IL-6, TNF-α, and TGF-β) were determined by ELISA, with a detection limit of 50 pg/mL (R&D Systems, Minneapolis, MN, United States).

Cells were plated at 1 × 10⁴ cells per well in 96-well plates and cultured for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cell culture medium to a final concentration of 250 μg/mL and incubated at 37 °C in a 5% CO₂ incubator for 4 h. Then, the medium was removed and formazan crystals were solubilized with 0.01 M HCl in isopropanol. Absorbance was measured at 595 nm in a plate spectrophotometer SpectraMax M (Molecular Devices, San Jose, CA, USA).

Concentration–response curves were generated to determine the optimal concentration and exposure time for CUR (BIOVEA CURCUMIN bcm-95®), EXT, Me08, and Me23 (Kindly donated by Dr Mirela Sairre, Federal University of ABC, Brazil). SH-SY5Y cells were cultured in 96-well plates (5 × 10⁴ cells/well) in 120 µl of cDMEM. Subsequently, the cells were treated with CUR at concentrations of 2.88 × 10^–2 µg/mL, 1.44 × 10^–1 µg/mL, 7.2 × 10^–1 µg/mL, 3.6 µg/mL, 18 µg/mL, and 90 µg/mL for 24 h. EXT was used at concentrations of 1.44 × 10^–1 µg/mL, 7.2 × 10^–1 µg/mL, 3.6 µg/mL, 18 µg/mL, and 90 µg/mL, while Me08 and Me23 were tested at concentrations of 7.5 µM, 15 µM, 30 µM, 60 µM, and 90 µM for 24 h. After the treatment, cell viability was assessed using a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA). Treated cells were incubated in 120 µl of DMEM supplemented with 10% FBS and 12 µl of 5 mg/ml MTT at 37 °C for 3 h. The formazan crystals formed were dissolved in 100 µl of dimethyl sulfoxide (DMSO). Optical density was measured at 590 nm in the spectrophotometer Spectramax M (Molecular Devices).

A375-KRAB infected with the empty lentivirus, enhancer-inhibited A375-KRAB, SK-MEL-28-KRAB infected with the empty lentivirus, enhancer-inhibited SK-MEL-28-KRAB, SK-MEL-2-KRAB infected with the empty lentivirus, and enhancer-inhibited SK-MEL-2-KRAB cells were separately seeded 100 µl into 96-well microplates with 3000 cells per chamber, and four replicates were performed for each group. After 24 h, culture medium with 1 or 2 µM vemurafenib or no treatment was added into each chamber. Cells were further incubated for 24, 48, 60, and 72 h, and cell viability was detected using the CCK-8 Cell Counting Kit (Vazyme, A311-01) according to the manufacturer’s description. Briefly, the original culture medium in the 96-well microplates was discarded, 100 µl of 10% CCK-8-DMEM high glucose solution was added into each chamber, and then the microplates were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm wave length by a microplate reader (Molecular devices, SpectraMax M). Viability was calculated as a percentage of control (A375-KRAB, SK-MEL-28-KRAB, or SK-MEL-2-KRAB cells without treatment) after background subtraction.