For measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity, liver samples (~ 10 mg) were homogenized in ice-cold assay buffer. Tissue homogenates were mixed with assay buffer, enzyme mix, developer, and substrate, incubated 60 min at 37 °C, and then measured in a microplate spectrophotometer (DYNEX, Chantilly, VA, USA). Hepatic enzyme activity was calculated following manufacturer’s instructions (BioVision, Milpitas, CA, USA, catalog#K752 and 753) according to the manufacturer’s instructions [20 (link)].
Microplate spectrophotometer
Manufactured by Dynex
Sourced in United States
The Microplate Spectrophotometer is a laboratory instrument used to measure the absorbance or optical density of samples in microplates. It provides accurate and reliable measurements of various biochemical and cell-based assays.
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Lab products found in correlation
5 protocols using microplate spectrophotometer
1
Measurement of Hepatic Enzymes: AST and ALT
2
Caspase 3 Activity Assay
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The generation of p-nitroaniline (pNA) from the caspase tetrapeptide substrate Ac-DEVD-pNA (Beyotime Institute of Biotechnology) was used as an index of caspase 3 activity, as described previously (16 (link)). Optical density of free pNA was measured at an absorption wavelength of 405 nm using a microplate spectrophotometer (Dynex Technologies, Inc., Chantilly, VA, USA).
3
Caspase-3 Activity Assay Protocol
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Caspase-3 activity was detected with a caspase assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, 2×106 cells were lysed with 100 µl lysis buffer on ice for 15 min and the cell lysates were then centrifuged at 16,000 × g for 10 min at 4°C. Reaction buffer (80 µl) and acetyl-DEVD-p-nitroanilin (pNA; 10 µl) was mixed with 10 µl lysate and the mixture was then incubated for 2 h at 37°C. The optical density of free pNA was measured at an absorption wavelength of 405 nm using a microplate spectrophotometer (Dynex Technologies, Chantilly, VA, USA). Caspase-3 activity was expressed as the relative absorbance ratio of samples vs. control group.
4
Cell Proliferation Assay with S1P
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The cells were seeded in a 96-well plate for 24 h, and treated with S1P or PET solution (as a vehicle control) for an additional 72 h. The medium was changed every 48 h. After the incubation, 50 μf of MTS (5 mg/mL) was added to the medium, and the plates were incubated for an additional 2 h. The optical density was then measured at 490 nm using a microplate spectrophotometer (Dynex technologies, Sullyfield, VA).
5
Cell Proliferation Assay Using CCK-8
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To assay for cell proliferation, Hep3B cell suspension of 100 μl were plated at a density of 1 × 104 per well in 96-well plates and serum starved for 24 h. 10 μl Cell Counting Kit-8 solution (Sigma-Aldrich) was added to each well of the plate under various treatment conditions. The absorbance was measured at 450 nm using a microplate spectrophotometer (Dynex Technologies Inc., Chantilly, VA, USA).
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