Caspase-3 activity was measured by a fluorimetric assay kit (Sigma-Aldrich) following the manufacturer’s instructions. Briefly, cell lysates were incubated for 1 h at 37 °C, in a solution containing caspase-3 substrate acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC). The hydrolysis of the Ac-DEVD-AMC by caspase-3 resulted by the release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety. The release of fluorescent AMC, index of caspase-3 activity, was assessed by a spectrofluorimeter (PerkinElmer Inc. USA) at 360 nm (excitation) and 460 nm (emission).
Fluorimetric assay kit
Manufactured by Merck Group
Sourced in United States
The Fluorimetric assay kit is a laboratory equipment product designed to quantify the presence and concentration of specific target analytes in a sample through fluorescence-based detection. The kit provides the necessary reagents and protocols to perform this type of analysis, but does not include interpretation or recommendations on its intended use.
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2 protocols using fluorimetric assay kit
1
Caspase-3 Activity Measurement by Fluorimetry
2
Caspase-3 Enzyme Activity Assay
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The caspase-3 enzyme activity was estimated by fluorimetric assay kit (Sigma-Aldrich, USA). Jurkat E6-1 cells (4 × 105) were treated with T. terrestris alkaloids extract at 70.20 μg and 140.40 μg concentrations per mL for 12 h and 24 h respectively. Vehicle and positive controls were taken as DMSO (0.1%) and Staurosporine (5 μM), respectively. After the incubation period, cells were lysed and the enzyme activity was estimated according to manufacturer's instructions. Caspase-3 activity was represented in pmol min−1 mL−1.
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