Life science uv vis spectrophotometer

Cortical neurons viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, M2128) reduction assay, which is an index of mitochondrial dehydrogenase activity. MTT (60 μg/mL) was added at the end of treatments and incubated for 1 h at 37 °C. The medium was removed, and 1 mL isopropanol-HCl was added to solubilize the resulting formazan salt. Formazan absorbance was determined at 570 nm in a Beckman Coulter life science UV/Vis spectrophotometer. Data are expressed as percent of MTT reduction relative to control.

Culture medium was withdrawn, and lactate dehydrogenase (LDH) activity was determined following the decrease in the optical density resulting from the oxidation of NADH using Pyruvate as a substrate. At the end of treatments, 200 μL of culture medium was collected and incubated with 9.4 mM NADH in 1 mM K₂HPO₄/KH₂PO₄ buffer for 5 min at room temperature. Pyruvate (20 mM) (Sigma-Aldrich, P-2256) was used to start the reaction, and the change in optical density at 340 nm was followed after 5 min in a Beckman Coulter life science UV/Vis spectrophotometer. Data are expressed as percent LDH release relative to control.

Cell viability was also measured by the LDH assay. Cells were exposed to 2 h GD and 15 h after GR, 200 μl of culture medium were collected for LDH activity determination. Samples were incubated with NADH 9.4 mm in K₂HPO₄/KH₂PO₄ 1 mm buffer during 5 min at room temperature. The reaction was started with pyruvate 20 mm and was followed for 5 min by the decrease in NADH fluorescence at 340 nm using a Beckman Coulter life science UV/Vis Spectrophotometer. Data were normalized to control values and are expressed as percent activity in the medium relative to the control. Cell viability was also determined by the Live/Death kit as described.^{30 (link)}