The enzymatic activities of HK, PFK and PK were assayed according to the instructions of the commercial kits (BC0745, Solarbio; BC0535, Solarbio; and BC0545, Solarbio). Cells or tissues were collected into centrifuge tubes and extracts were added in appropriate proportions. Cells were crushed with ultrasound (Scientz, Scientz-IID) and tissues were homogenized in ice bath. The extracts were then centrifuged at 8000 g for 10 min at 4°C. The supernatant was taken for testing. The reagents were added sequentially to the 96-well plate according to the instructions. After mixing well, the absorbance difference values were measured at different times on a microplate reader (MuitisKan, ThermoFisher) according to the instructions. The wavelength of the microplate reader was set to 340 nm.
Muitiskan
Manufactured by Thermo Fisher Scientific
The MultisKan is a microplate reader designed for a variety of absorbance-based assays. It features a xenon flash lamp light source and a monochromator for wavelength selection. The instrument can read 6- to 384-well microplates and supports multiple measurement modes, including endpoint, kinetic, and spectral scanning.
Automatically generated - may contain errors
4 protocols using muitiskan
1
Enzymatic Activity Assays for Glycolytic Enzymes
2
Glutathione Quantification Protocol
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No less than 5 million cells were collected according to glutathione determination instructions (BC1175, Solarbio). The cells were washed with PBS and then resuspended with 1 mL reagent A. The cells were ultrasonically broken (Scientz, Scientz-iID) and centrifuged. Then, supernatant measurements were collected. The wavelength was adjusted to 412 nm with an enzyme marker (MuitisKan, ThermoFisher). Finally, the GSH content was calculated according to the number of cells.
3
Cell Viability Assay using Thiazolyl Blue
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The thiazolyl blue powder (IM0280, Solarbio) was dissolved in PBS (P1020, Solarbio) and stored in the dark at 4°C. HT22 cells were plated at 2000 cells/well in a 96-well plate in a volume of 100 μL. Following drug intervention for 24 h, HT22 cells were given 20 μL of thiazolyl blue solution. The 96-well plate was placed in a 37°C incubator and incubated in the dark for 4 h. After aspirating the medium, 100 μL of DMSO was added to dissolve the purple precipitate at the bottom. Finally, absorbance was determined at a wavelength of 490 nm using a microplate reader (MuitisKan, ThermoFisher).
4
Quantifying Pyruvate in HT22 Cells
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Pyruvate content was measured according to the manufacturer’s instructions (BC2200, Solarbio). HT22 cells were collected into a centrifuge tube, and the supernatant was discarded after centrifugation (200 g, 5 min). The extract was added in appropriate proportions and crushed by ultrasound (Scientz, Scientz-IID). Hippocampal tissue was added to the extract according to the ratio of about 0.1 g tissue to 1mL extract and homogenized in an ice bath. The solution was allowed to stand for 30 min, centrifuged at 8000 g for 10 min at room temperature, and the supernatant was taken to be measured. Reagent II was added after standard solution or sample was mixed with reagent I in a 96-well plate, and absorbance A was determined at a wavelength of 520 nm using a microplate reader (MuitisKan, ThermoFisher). Finally, the pyruvate content was calculated after measuring the protein concentration of the samples using the BCA method.
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