DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet.
Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.
Lee
Brian W
BW
Immunochemistry Technologies, LLC, Bloomington, MN, USA. brian@immunochemistry.com
Johnson
Gary L
GL
Hed
Sally A
SA
Darzynkiewicz
Zbigniew
Z
Talhouk
Jamil W
JW
Mehrotra
Sanjiv
S
D017209:Apoptosis; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology*; D002452:Cell Count / Q000379:methods*; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods*; D009842:Oligopeptides; D010078:Oxazines; D010447:Peptide Hydrolases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014018:Tissue Distribution
C487862:(aspartyl-glutamyl-valyl-aspartate)2-cresyl violet; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010078:Oxazines; D010447:Peptide Hydrolases; C108711:DEVDase