Experiments on biochemical analysis of cell death

Summary

This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Colorimetric quantification of caspase activation

Materials and Instruments

Cells
PBS Eppendorf DMSO
Microtitre plates

Move

1. 250 g centrifugation for 5 minutes to collect ^2X106~5X106 cells, wash once with cold PBS of pH 7.2, and resuspend with 200 μl of lysis buffer.

2. Incubate cells on ice for 30 minutes.

3. centrifuge the samples at 13000 r/min for 5 min with Eppendorf and take the top sample for caspase activity assay: reserve one aliquot of each sample for protein quantification.

4. Dissolve the substrate in water or DMSO to make a 20 mmol/L storage solution that can be stored at -20°C protected from light.

5. Place the reaction mixture in a microtitre plate and read the reaction data on a SpectraMax microtitre plate analyzer with a kinetic device under a 405 nm filter. Determine the initial rate of hydrolysis of the substrate by testing the sample every 2-20 seconds; ensure that the resulting rate of hydrolysis of the substrate is linear.

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