Labsystems multiskan mcc 340

Toxicity of NPs was assessed as previously published [57 (link)]. Briefly, 661W-A11 cells were cultured in 96-well plates at a density of 6000 cells/well and increasing dilutions of NPs were added to the culture medium. After 48 or 72 h, the medium was aspirated and the cells in each well were incubated for 2 h at 37 °C with 50 µL of 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) diluted in the culture medium. The supernatant was removed and the purple formazan crystals were dissolved in 100 µL of isopropanol and shaken for 10 min. Lastly, the optical density (OD) was measured at 570 nm using a microplate reader (Labsystems Multiskan MCC/340, Fisher Scientific, Rodano, Italy).

bEnd.3 cells (2 × 10⁵ cells/mL) were seeded in 98-well plates to monitor all experiment conditions, including pretreatment, OGD injury, and reperfusion. Next, cells were rinsed twice with phosphate-buffered saline (PBS), and culture medium was replaced with serum-free medium and 100 μL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) (Sigma, MO, USA) solution (5 mg/mL in PBS) per well. After 1 h of incubation, medium was removed and dimethyl sulfoxide (DMSO) was added to solubilize the purple formazan product of MTT treatment. The supernatant from each well was analyzed using an ELISA plate reader (Labsystems Multiskan MCC/340; Fisher Scientific, PA, USA) at a wavelength of 570 nm, with background subtraction at 650 nm. All experiments were repeated at least three times. Cell viability in the control medium, without any treatment, was represented as 100%. Cell viability was reported as a relative value, compared to the control group.

SH-SY5Y cells (2 × 10⁵ cells/mL) were seeded in 96-well plates for monitoring all experimental conditions including melatonin pretreatment (100 μM) and insulin stimulation (1 μM), separately. Next, culture medium was replaced with serum-free medium, and 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St.Louis, MO, USA) solution (5 mg/mL in PBS) was added to each well. After incubation for 1 h, medium was removed and dimethyl sulfoxide was added to each well to solubilize the purple formazan product of MTT reaction. The supernatant from each well was analyzed using a microplate reader at 570 nm (Labsystems Multiskan MCC/340; Fisher Scientific, Pittsburgh, PA, USA). All experiments were repeated three times. Cell viability in medium of non-treated cells was considered 100%.