Multiscan spectrophotometer

Determination of the concentration of carbohydrate metabolism metabolites was carried out calorimetrically using the UV-mini 1240 spectrophotometer, Shimadzu. For analysis of glucose concentration, a set of reagents Climates-GOT from SPC “Eco-Service”, Moscow, Russia (Cat No. B-11061) was used. The concentration of lactate was determined using the set from OOO «Olvex Diagnosticum», Russia (Cat. No. 019.002). The activity of glucose-6-phosphate dehydrogenase was determined using the set of reagents «Sentinel», Milano, Italy (Cat. No. 17005) on the biochemical analyzer StatFaks 04, the USA. The activity of the regulatory enzyme of glycolysis, hexokinase, was determined by staining the probe using the set of reagents from «Sigma-Aldrich» (St. Louis, MO, USA) (Cat. No. MAK091) on the MULTISCAN spectrophotometer (Thermo Scientific, Vantaa, Finland). The immunoenzyme method of «sandwich» type was used to determine the amount of transketolase (Human SimpleSep ELISA Kit “Abcam”, Waltham, MA, USA; Cat. No. ab187398) and glycogen synthase kinase 3β, (ELISA kit “Puda Scientific”, WUHAN, China; cat. No. PD-H7862E); optical density was registered at 450 nm on the MULTISCAN spectrophotometer (Thermo Scientific, Vantaa, Finland).
The results were corrected to 1 g of protein which was determined by the Lowry method with a set of reagents from OOO «Firma Syntacon»,Saint Petersburg, Russia.

Cell viability was assessed by using the Colorimetric (MTT) Kit for Cell Survival and Proliferation (Merck Kft.; Darmstadt, Germany) according to the manufacturer’s instructions. MTT-derived formazan was measured at 530 nm test and 630 nm reference wavelengths in a multiscan spectrophotometer (Thermo Fisher Scientific; Waltham, MA, USA). Cell viability was expressed as the percentage of viable cells in the total cell population.
Apoptotic and necrotic cells were detected by using Annexin-V-FLUOS Staining Kit (Roche; Basel, Switzerland) and fluorescence microscopy according to the manufacturer’s instructions. Cells with green fluorescence (Annexin V labeling) were considered as apoptotic while those with red or both green and red fluorescence (propidium iodide DNA staining) were considered as necrotic. A minimum of 1000 cells was counted in each experimental condition. Apoptosis index was calculated as (number of apoptotic cells) / (number of all cells counted) × 100.

MTT was used to measure cell viability. First, cells were seeded in 96-well plates with 5000 cells/well and incubated at 37 °C for 24 h before treatment with different concentrations of EN for different times. Then, 50 μL of MTT (2.5 mg/mL dissolved in PBS) solution was added into each well for a 2 h incubation. Finally, the medium was carefully replaced with 200 μL of DMSO. After shaking for 3 min, we measured the absorbance at 570 nm with a multiscan spectrophotometer (Thermo Scientific). The relative cell viability rate = (the average absorbance of the experimental group/the average absorbance of the control group) × 100%. All data were repeated three times.
Cell viability assay was also evaluated using a CCK8 assay kit. After the indicated treatment, 10 μL of CCK-8 was added to each well, and the cells were subsequently incubated at 37 °C for 1~4 h. The absorbance was measured at 450 nm using the microplate reader.