Caspase-3/7 Assay: A Luminescence-Based Assay to Quantify Apoptosis by Measuring Caspase-3/7 Activities in Frog Skin Explants

Abstract

Source: Brannelly, L. A. et al., Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis. J. Vis. Exp. (2018).

This video describes an in vitro Caspase-3/7 luminescent assay to quantify cell death or apoptosis. The assay measures luminescence produced following caspase-3/7 cleavage of a pro-luminescent DEVD substrate. The luminescence generated is proportional to caspase-3/7 activity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animal Husbandry and Monitoring

1. House animals individually, in an environment appropriate for the species, with an appropriate water, feeding, and cleaning schedule. Check animals daily.
   1. Use adult individuals of the threatened Litoria verreauxii alpina (for the Caspase 3/7 assay, Section 4), donated from captive breeding facilities. Maintain the animals at 15 - 18 °C, on a moss and gravel substrate, mist them daily with reve....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
POLARstar Omega	BMG Labtech		Luminescent plate reader
384 well flat clear bottom plate	Corning	3707
384 well low flange white flat bottom plate	Corning	3570
Agar Bacteriological (Oxoid)	Fisher	OXLP0011B
Lactose Broth (Oxoid)	Fisher	OXCM0137B
Sodium Bicarbonate	Fisher	BP328-500
Tricane-S (MS-222)	Fisher	NC0872873
Tryptone	Fisher	BP1421-500
Bovine Serum Albumin	Invitrogen	15561020
Sterile rayon swab	Medical Wire & Equipment	MW-113
Coomassie Bradford reagent	Pierce	23200
Caspase Glo 3/7	Promega	G8090
HEPES buffer	Sigma-Aldrich	H0887-20ML
Magnesium chloride	Sigma-Aldrich	1374248-1G
Gelatin hydrolysate Enzymatic	Sigma-Aldrich	G0262
PBS (Phosphate Buffered Saline), pH 7.2 (1X)	Thermo/Life	20-012-043
Prepman	Thermo/Life	4318930
TaqMan Fast Advanced Master Mix	ThermoFisher	4444556
Clorox bleach	Clorox
Ethanol, 200 Proof, Molecular Grade	Fisher	BP2818500
ZEISS Axio Scan florescent miscroscope	Carl Zeiss		Florescent microscope
3.2mm stainless steel beads	BioSpec	11079132SS
Primer ITSI-3 Chytr (5′-CCTTGAT ATAATACAGTGTGCCATATGTC-3′)	Taqman		Individual design for primers and probe
Primer 5.8S Chytr (5′-TCGGTT CTCTAGGCAACAGTTT-3′)	Taqman		Individual design for primers and probe
Minor groove binder probe Chytr MGB2(5′-CGAGTCGAAC-3′)	Taqman		Individual design for primers and probe
Rotor-Gene qPCR Instruments	Qiagen		qPCR machine
Microcentrifuge tubes 1.5ml	Fisher	02-681-372
Cell culture petri plates	Nunc	263991
Mini-beadBeater Zircornia-Silicate Beads, 0.5mm	BioSpec	11079105Z