Caspase 3/7 (DEVD-FMK)
Label: Green (#23101), Ultrabright Green (#13101) Fluorescence
Size: 100 tests
Format: in vitro
Storage: -20° C
The CAS-MAP™ in vitro Caspase 3 apoptosis assay utilizes the caspase family inhibitor DEVD-FMK conjugated to a fluorescent label as a marker for the detection of Caspase-3/7 in cell based assays. DEVD-FMK is cell permeable, nontoxic, and irreversibly binds to activated caspases in apoptotic cells. Any unbound reagent diffuses out of the cell and is washed away.
The ProBo™ ultrabright green fluorescent assay (#13101) is up to 17x brighter than traditional assays and less sensitive to photobleaching, which may be desirable for tissue microscopy studies.
Target: Caspase 3/7
Sample Type: whole cells or tissue
Assay Time: 30-60 minutes
Analysis: Flow cytometry, fluorescent microscopy, fluorescent plate reader
Excitation/Emission: 488/530 nm
DEVD-FMK, 2 vials
Wash Buffer, 1 PBS tablet
Instructions for Use
Cell Cultures: Culture cells as required to induce apoptosis. The density of the cultured cells should not exceed 106 cells/ml. Typically, 106 cells are aliquoted into wells of a 24 well culture plate in a final volume of 1 ml. Investigators must optimize the conditions and kinetics for induction of apoptosis in their system. A control group of cells not induced to undergo apoptosis should also be cultured. It is recommended that induced and non-induced cultures be set up for each labeling condition.
Sample Staining: Cells can be stained directly during the last 30 minutes of the apoptosis induction period. Add 10 µL of CAS-MAP per 1 mL culture volume. The staining is performed at 37° C. After the staining period, harvest the cells into 5 mL tubes, centrifuge at 500 x g for 5 minutes and wash once with 4 mL of 1X Wash Buffer (diluted from 10X Wash Buffer provided in kit) to remove unbound reagent. Re-suspend the cells in PBS for flow cytometry analysis.
Flow Cytometry Analysis: Observe induced and non-induced cells on a SS versus FS linear dot plot to identify and gate on cells of interest. Note that apoptotic cells have been described to undergo changes in their light scattering properties. Make sure that the scatter gate used to identify cells of interest will accommodate these changes in the light scattering properties of the cells.
Detection is usually collected on the FL1 channel. Generate histograms depicting FL1 (x-axis) versus cell count (y-axis). Monitor the fluorescence of the non-induced cell culture. Voltage adjustment of the photomultiplier tubes may be desirable to optimally position the fluorescence distribution around the first log decade of the histogram display. Mark the regions of the histogram that identify positive and negative events. Cells from other culture conditions may now be tested for the presence of apoptotic cells. The caspase positive cell population will appear as a separate peak or as a shoulder of the first peak demonstrating increased fluorescence intensity.
Sample staining with CAS-MAP in vitro products can be achieved during the last 15 to 60 minutes of the apoptosis induction culture period. Long incubation times with CAS-MAP products may inhibit activation of caspases and the apoptotic cascade. This could result in reduced CAS-MAP binding. Short incubation times with CAS-MAP during the staining process may not allow for the CAS-MAP binding reaction to go to completion.
CAS-MAP staining may also be carried out following cell harvest. Collect the cells in 5 mL (12 x 75 mm) tubes and wash once as described in Sample Staining. Re-suspend the cells in 1 mL of PBS. Add 10 µL of CAS-MAP to each tube and incubate for 30 minutes at 37° C. Wash cells once in PBS to remove unbound reagent. Re-suspend the cells in PBS for flow cytometry analysis.
Members of the caspase enzyme family (cysteine proteases with aspartate specificity) play significant roles in both inflammation and apoptosis. Caspases exhibit catalytic and substrate-recognition motifs that have been highly conserved. These characteristic amino acid sequences allow caspases to interact with both positive and negative regulators of their activity. The substrate preferences or specificities of individual caspases have been exploited for the development of peptides that successfully compete for caspase binding while maintaining their distinctive aspartate cleavage sites at the P1 position.
It is possible to generate reversible or irreversible inhibitors of caspase activation by coupling caspase-specific peptides to certain aldehyde, nitrile or ketone compounds. Fluoromethyl ketone (FMK)-derivatized peptides act as effective irreversible inhibitors with no added cytotoxic effects. Inhibitors synthesized with a benzyloxycarbonyl group (also known as a “Z” group) at the N-terminus and methyl esters exhibit enhanced cellular permeability. Conjugation of the inhibitor to afluorescent label allows for intracellular detection of the reagent upon binding to the active caspase enzymes. The detection of active caspases by flow cytometry is a rapid method for identifying cells undergoing apoptosis.