This article discusses the role of caspase-3 and caspase-7 in apoptosis and details a method for assessing their activity in frog toe skin explants. The protocol involves lysing cells, extracting proteins, and measuring luminescence to determine caspase activity.
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.
To extract proteins from a frozen frog toe sample, place the sample in a 1.5-milliliter screw cap microcentrifuge tube, containing 100 microliters of sample buffer and two stainless steel beads.
Use a bead beater at maximum speed to lyse the cells. After this, place the tube on ice for 3 minutes, then, centrifuge the tube to collect the supernatant.
To perform the caspase 3/7 assay in a 384-well plate, add 10 microliters of sample buffer as blank, and 10 microliters of protein extract in triplicate. Then, pipette 10 microliters of commercially available caspase 3/7 reagent to the protein extract and blank wells. Then, shake the plate gently for 15 seconds to mix the two reagents.
Incubate at room temperature and dark for 30 minutes. Finally, use a luminescent plate reader to measure the luminescence of the sample.
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