To assess induction of apoptosis, a colorimetric caspase 3 activity assay was employed according to the manufacturer’s protocol (Sigma). Briefly, cell lysates from both adherent and floating cells were centrifuged at 20000 × g for 10 min. Twenty μg of the supernatant was incubated with 85 μL of assay buffer plus 10 μL of caspase 3 substrate acetyl-Asp- Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) in a 96-well plate at 37 °C for 12 h. The samples were then read at 405 nm in an ELISA reader.
Caspase 3 activity assay
Manufactured by Merck Group
The Caspase-3 activity assay is a laboratory instrument designed to measure the activity of the enzyme Caspase-3. Caspase-3 is a key player in the process of apoptosis, or programmed cell death. The assay provides a quantitative assessment of Caspase-3 activity, which is useful for studying cell death processes in various biological systems.
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4 protocols using caspase 3 activity assay
1
Caspase 3 Apoptosis Assay Protocol
2
Apoptosis and Autophagy Assays
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Equal numbers of cells were plated and treated the following day by serum deprivation for 24 h or with 100 nM lapatinib (Santa Cruz) for 24 h prior to analysis by Caspase-3 activity assay (Sigma) or trypan blue exclusion and cell counting, which was performed using the cellometer mini cell counter (Nexcelom). For autophagy, cells were treated with 100 uM chloroquine (Sigma) for the indicated times and analyzed by Western blotting or microscopy (Nikon E800).
3
Caspase-3 Activity Measurement
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Equal numbers of cells (20,000 cells/well) were plated on 96-well dishes and treated the following day with STU for 24 hours prior to analysis by Caspase-3 activity assay (Sigma) as previously described [10 (link)].
4
Caspase-3 Activity Assay Protocol
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Equal numbers of cells (5,000 or 20,000 cells/well) were plated on 96-well dishes and treated the following day with indicated inhibitors for 24 hours prior to analysis by Caspase-3 activity assay (Sigma) as previously described [16 (link)].
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