Caspase 1, 4 Probe
Sequence: YVAD-FMK or WEHD-FMK
Label: Green (#21100), Ultrabright Green (#11100), Red Fluorescence (#11200)
Size: 10 mice
Format: in vivo
Storage: -20° C
CAS-MAP™ Caspase 1 probe utilizes the caspase family inhibitors YVAD-FMK or WEHD-FMK conjugated to a green or red fluorescent label. The Caspase-1 is cell permeable, nontoxic, and irreversibly binds to activated caspase 1 in tissue.
For studies looking at tissue microscopy, the ultrabright green may be desirable.
Target: Caspase 1, 4
Sample Type: in vivo (IV injection or intra-tumoral injection)
Imaging Time: 30-60 minutes post injection
Analysis: Live animal imaging, Flow cytometry, fluorescent microscopy
Expected Tissue Penetration:
Green Probe 2-3 mm
Red Probe 5-7 mm
Green Probe 488 nm/533 nm
Red Probe 660 nm/680-690 nm
Probe (Red-YVAD-FMK or Green-WEHD-FMK), 1 vial
Injection Buffer, 1 bottle
Instructions for Use:
Sample Protocol (Mouse Model):
EQUIPMENT: High grade anhydrous DMSO (50 μL) distilled, deionized (DD) sterile H2O (45 mL), syringe filter (0.2 μm), injector (syringe and needle), tissue extraction and imaging tools, fluorescence detection equipment (flow cytometer, plate reader, fluorescence microscope, or animal imager).
STEP 1: Expose animals to experimental condition, and create positive and negative controls.
STEP 2: Reconstitute vial of CAS-MAP in vivo with 50 μL DMSO to form stock concentrate.
STEP 3: Dilute injection buffer 1:10 with DD H2O and sterilize diluted injection buffer by filtration through a 0.2 μm syringe filter or equivalent.
STEP 4: Immediately prior to injection, add 550 μL of sterile 1X injection buffer per vial to form a 1X CAS-MAP in vivo solution. Inject product within 1 hour of dilution; protect from light during handling.
STEP 5: Inject 100 µL of the 1X CAS-MAP in vivo solution into the tail vein (or other desired intravenous injection site) of each mouse.
STEP 6: Once injected, let circulate for 60 minutes.
STEP 7: Examine tissues under a fluorescent microscope, examine live animal via animal imager, or sacrifice and excise cells for analysis via flow cytometer or plate reader.
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. Fluoro-methyl ketone (FMK)-derivatized peptides act as effective irreversible inhibitors with no added cytotoxic effects. Inhibitors synthesized with a benzyloxy-carbonyl group (also known as a “Z” group) at the N-terminus and methyl esters exhibit enhanced cellular permeability. Conjugation of the inhibitor to a fluorescent label allows for intracellular detection of the reagent upon binding to the active caspase enzymes.
Activation of caspases plays a central role in apoptosis. Apoptotic cells have more active caspases than control cells, and therefore fluoresce brighter when labeled with CAS-MAP™. The fluorescent signal resulting from the bound CAS-MAP™ probe provides a direct measure of active caspase 1 at the time of injection, and can be readily analyzed via flow cytometry or fluorescence microscopy.
CAS-MAP™ in vivo probe eliminates false positive bias associated with ex vivo processing, and provides a true representation of in vivo active caspase measurement as a result of the experimental condition.