Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants
Transcripción
Caspase-3 and caspase-7 are proteases that recognize the DEVD tetrapeptide sequence in target proteins, cleaving them at the C-terminus of the aspartic acid residue. These play a prominent role in the apoptosis – programmed cell death – cascade.
To assess caspase-3/7 activity in apoptotic cells, begin with a tube containing a frog toe skin explant infected with apoptosis-inducing fungi. Add a suitable buffer to the tube and a few metallic beads.
Use bead-beating to disrupt the skin cells and release cellular contents, including caspase-3/7 proteins. Centrifuge the tube, pelleting cell debris, and organelles. Aspirate the protein-rich supernatant from the tube and pipette into the wells of a multiwell assay plate.
Supplement the wells with the luminogenic caspase reagent. This reagent consists of a proluminescent substrate containing the DEVD peptide in a buffered solution containing ATP, magnesium ions, and luciferase enzyme.
Mix the contents of the well and incubate. During incubation, caspase-3/7 cleaves the proluminescent substrate, releasing aminoluciferin – a substrate for luciferase – into the solution.
In the presence of ATP and magnesium ions, luciferase oxidizes the released aminoluciferin, generating a bioluminescent product. The amount of light produced during the reaction is proportional to the caspase activity.
