Green FLICA™ Caspase 1 Assay Kit

Measure Caspase-1 Activity in Whole, Living Cells
Catalog no. 97 (25 tests), $179 USD
Catalog no. 98 (100 tests)

Caspase 1 (ICE)
Members of the mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation. Originally identified as Interleukin -1β Converting Enzyme or ICE (1, 2), caspase 1 along with caspases 4, 5, and 12 comprise the inflammatory subfamily of caspase enzymes (3). In rodents, caspase 11 is also an inflammatory caspase (3). Caspase 1-mediated cleavage of pro-IL-1β results in the biologically active form of this critical immune response regulator. Caspase 1 has also been found to play a role in processing a wide variety proteins; most notably several cytokines (4-6) and enzymes within the glycolytic pathway (7). Finally, the creation of the inflammasome during host responses to pathogens leads to the activation of caspase 1 (8, 9). Inflammation-related disease models have illustrated a role for caspase 1 in asthma, rheumatoid arthritis, multiple sclerosis and other disorders (8, 9).

Like other caspase family members, caspase 1 is a heterodimer comprised of two subunits, 20 kDa and 10 kDa in size (10, 11). Caspase 1 is autocatalytically activated following oligomerization. Active caspase enzymes exhibit catalytic and substrate specificities comprised of short tetra-peptide amino acid sequences that must contain an aspartate in the P1 position (12 - 14). These preferred tetra-peptide sequences have been used to derive peptides that specifically compete for caspase binding (15 - 17). In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases typically require four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue (14). Most inflammatory caspases prefer a hydrophobic amino acid such as tyrosine or tryptophan in the P4 position (14). Addition of a fluoromethyl ketone (FMK) to the tetrapeptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme (18). Furthermore, conjugation of a fluorescent moiety at the amino terminus yields a probe that allows for the detection of caspase 1 activity (19 - 21).

FLICA™ Caspase 1 Detection Mechanism
The FLICA™ reagent FAM-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the FAM-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-YVAD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active caspase 1 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA™ can be analyzed by 96-well-plate based fluorometry, fluorescence microscopy, or flow cytometry. The carboxyfluorescein (FAM) FLICA™ reagent has an optimal excitation range from 490 - 495 nm and optimal emission range from 515 - 525 nm. Cells labeled with the FLICA™ reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may also be analyzed with propidium iodide or Hoechst stain to detect necrosis or changes in nuclear morphology respectively.

Additional caspase-1 probes are in development. To receive pre-release information about ICT's future caspase-1 probes, please join our newsletter list and indicate your area of interest.

1. Black, R.A., Kronheim, S.R., Merriam, J.E., March, C.J., and Hopp, T.P. (1989) A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta. J. Biol. Chem. 264:5323-26.
2. Kostura, M.J. et.al. (1989) Identification of a monocyte specific pre-interleukin 1 beta convertase activity. PNAS USA 86:5227-31.
3. Scott, A. M., and M. Saleh. (2007). The inflammatory caspases: guardians against infections and sepsis. Cell Death Differ. 14:23-31.
4. Ghayur, T., et.al. (1997). Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386:619-23.
5. Schmitz, J., et al., (2005) IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 23:479-90.
6. Kumar, S. et. al. (2002) Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL1F7B binds to the IL-18 receptor but does not induce IFN-gamma production. Cytokine 18:61-71.
7. Shao, W. et al., (2007) The caspase-1 digestome identifies the glycolysis pathway as a target during infection and septic shock. J. Biol. Chem. 282:36321-29.
8. McIntire, C. et al, (2009) Inflammasomes in infection and inflammation. Apoptosis 14:522-35.
9. Franchi, L. et al. (2009) The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nature Immunol. 10:241-47.
10. Thornberry, N.A., et al. (1992) A novel herterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356:768-74.
11. Ayala, J.M., et al. (1994) IL-1beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor. J. Immunol. 153:2592-99.
12. Cryns, V., and Yuan, J. (1998) Proteases to die for. Genes Dev. 12:1551-1570.
13. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D., and Wong, W.W. (1997) Substrate specificities of caspase family proteases. J. Biol. Chem. 272:9677-9682.
14. Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., and Thornberry, N.A. (1998) Inhibition of human caspases by peptide-based macromolecular inhibitors. J. Biol. Chem. 273:32608-32613.
15. Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22:8543-8567.
16. Nicholson, D.W. (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6:1028-1042.
17. Thornberry, N.A., and Lazebnik, Y. (1998) Caspases: enemies within. Science 281:1312-1316.
18. Rauber, P., Angliker, H., Walker, B., and Shaw, E. (1986) The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteases and cysteine proteinases. Biochem. J. 239:633-640.
19. Bedner, E., Smolewski, P., Amstas, P., and Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res. 259:308-313.
20. Amstad, P.A., Yu, G., Johnson, G.L., Lee, B.W., Dhawan, S., and Phelps, D.J. (2001) Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques 31:608-610.
21. Smolewski, P., Bedner, E., Du, L., Hsieh, T.C., Wu, J.M., Phelps, D.J., and Darzynkiewicz, Z. (2001) Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44:73-82.

Sample Protocol:

FLICA™, Fluorescent-Labeled Inhibitor of Caspases, is a simple yet accurate method to measure apoptosis via caspase activity in whole cells. Four sample protocols are outlined below. 

Suspension Cells

1. Culture your cells up to 1 x 10⁶ cells/mL.
2. Follow experimental protocol where caspase activity will be investigated; create positive and negative controls for caspase activity.
3. Reconstitute the reagent with 50µL DMSO to form the stock concentrate (can be frozen for future use).
4. Dilute the stock concentrate with 200µL 1X PBS to form the working solution.
5. Add ~10µL of the working solution directly to a 300-500µL aliquot of your cell culture for labeling.
6. Incubate 30 minutes -1 hour.
7. Wash and spin cells two or three times, or let incubate for 1 hour with fresh media or 1x "apoptosis wash buffer" (#634 or 635, included in kit).
8. If desired, label cells with Hoechst stain.
9. If desired, label cells with Propidium Iodide or 7-AAD.
10. If desired, fix cells.
11. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

Frozen Tissues

1. Prepare frozen tissues according to the experiment.
2. Allow slides to air-dry.
3. Fix slides with acetone for 1 minute.
4. Rehydrate slides by washing (twice for 5 min) in TBS-tween (TBSt) or PBS-tween (PBSt).
5. Block slides for 20 minutes (such as 20% Aquablock in media with 0.2% tween).
6. Dilute 150X FLICA stock 1:50 in PBS to form a 3X working solution. For example, add 50 µL 150X stock to 2450 µL PBS (2.5 mL total).
7. Add 50 µL of 3X FLICA™ and incubate >1hr protected from light.
8. Wash with TBSt or PBSt (twice for 5 min) by setting slides in slide incubation dish containing 1X wash buffer.
9. Develop with DAPI and coverslip.
10. Store samples at 2-8°C for short term storage, staining will last at -20° C for long periods.

Adherent Cells

Adherent cells need to be carefully washed to avoid the loss of any cells which round up and come off the plate surface. Loose cells may be harvested from the plate or slide surface and treated as suspension cells, while those remaining adherent to the surface should be washed as adherent cells. If the adherent cells are trypsinized, the loose cells can be recombined with the trypsinzed pool, or the washed loose cells can then be recombined with the adherent portion when the analysis is performed. If growing adherent cells on a tissue culture plate, the entire plate may be gently spun as part of the wash process to sediment any loose floating cells.  Avoid any attempts to trypsinize cells prior to labeling with a vital dye such as PI.  Trypsin-exposed cell membranes could become transiently permeant to vital dyes such as PI for a variable time period, depending upon the cell line. Cells may be labeled with FLICA™ before or after trypsinization.

Adherent Cells: Trypsinization prior to FLICA™ labeling and FACS analysis:

1. Culture cells in T25 flasks and expose to the experimental conditions.
2. Apoptotic cells may detach and begin to float into the media. Save and spin to pellet and include these cells in your analysis.
3. Trypsinize adherent cells; neutralize with trypsin inhibitor present in 20% FBS-cell culture media; pool cells with any pellets created in #2; add a few mL media.
4. Spin ~5 minutes at 220 x g and remove all but ~100 µL supernatant.
5. Count cells and adjust volume of cell suspension to fit the experiment (typically 300-500 µL). Transfer cells into a 15 mL tube.
6. Add 10-17 µL of 30X FLICA.
7. Incubate at 37°C, 30-60 minutes, mixing gently every 10 minutes.
8. Wash by adding ~10mL media and incubate at 37°C for 60 minutes to allow any unbound FLICA™ to diffuse out of the cells.
9. Spin at 220 x g for 5 minutes; aspirate supernatant.
10. Add ~300µL 1X "apoptosis wash buffer". Put cells on ice, and protect from light.
11. If desired, add 30 µL fixative.
12. Analyze cells with a flow cytometer.

Adherent Cells: FLICA™ label prior to trypsinizing, and FACS analysis:

1. Seed 5-8 x 10⁴ cells in a 24-well plate in a final volume of 600 µL and let attach for 24 hours.
2. Expose cells to the experimental conditions.
3. Add 1-4 µL of FLICA™ 150X stock concentrate and incubate 1-3 hours at 37°C.
4. Remove supernatant containing any rounded up cells and set aside in labeled tube.
5. Wash adherent cell monolayer by gently adding PBS to cover the adherent cell monolayer.
6. Remove PBS and combine with cells previously set aside in step 4.
7. Add trypsin – versene to barely cover the attached cell monolayer. 
8. Allow cells to detach and remove detached cells by adding 1 mL of cell culture media + 20% FBS to the trypsinized cells in the wells.   
9. Add detached cells from the trypsinization step to supernatant from step 4.
10. Add 2 mL of cell culture media + 20% FBS to each tube containing trypsinized cells.
11. Spin cells at 220 x g for 5 min. Remove supernatant and discard. Add 1mL 1x "apoptosis wash buffer."
12. Spin cells at 220 x g for 5 min. Remove supernatant. Add 1mL 1x "apoptosis wash buffer."
13. Spin cells at 220 x g for 5 min. Remove supernatant and resuspend in 300 µL 1X "apoptosis wash buffer."
14. If desired, add 30µL fixative.
15. Analyze on FACS immediately.

1. Grabarek, J., P. Amstad, and Z. Darzynkiewicz. 2002. Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases. Human Cell 15(1):1-12.
2. Grabarek, J., and Z. Darzynkiewicz. 2002. In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors. Exp. Hematol. 30:982-989.
3. Grunewald, S., Paasch, U., Said, T.M., Sharma, R.K., Glander, H.J., Agarwal, A. 2005. Caspase activity in human spermatozoa in response to physiological and pathological stimuli. Fertil. Steril. 83:1106-1112.
4. Scotton, C.J., Martinez, F.O., Smelt, M.J., Sironi, M., Locati, M., Mantovani, A., and Sozzani, S. 2005. Transcriptional profiling reveals complex regulation of the monocyte IL-1β system by IL-13. J. Immunol., 174: 834-845.
5. Bauernfeind, F. et al. 2009. NF-B Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression. J. Immunol. 183: 787-791.
6. Martin, U. et al. 2009. Externalization of the Leaderless Cytokine IL-1F6 Occurs in Response to Lipopolysaccharide/ATP Activation of Transduced Bone Marrow Macrophages. J. Immunol., 183: 4021-4030.
7. Abdul-Sater et al. 2009. Inflammasome-Dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia Trachomatis. J. Biol. Chem 284: 26789-26796.
8. Knodler, et al. 2010. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. PNAS, 107: 17733-17738.
9. Abdul-Sater et al. 2010. Enhancement of Reactive Oxygen Species Production and Chlamydial Infection by the Mitochondrial Nod-like Family Member NLRX1. J. Biol. Chem., 285: 41637 - 41645.

 Suspension cells were treated with an apoptosis-inducing agent or DMSO, a negative control, for 4 hours, washed twice, then incubated with ICT’s green caspase 1 inhibitor probe, FAM-YVAD-FMK, for 1 hour and examined under a fluorescence microscope. The top images reveal several experimental cells, all of which fluoresce green, therefore they have active caspase 1. The lower brightfield image at right reveals many control cells in the field of view, however the corresponding fluorescence image is dark (lower left); none of these cells have active caspase 1 (Dr. Brian W. Lee, ICT).

Do you have interesting FLICA data to share?  Contact us with your data and feedback, and it could be featured in our blog or newsletter!

How many tests can be run with the trial size and regular size kits?
The trial size FLICA kit provides enough reagent to test 7.5mL of cell culture samples - approximately 25 tests. The regular size FLICA kit provides reagent for testing 30mL of cell culture samples - approximately 100 tests.

What is one "test"?
One "test" is a 300uL aliquot of cells grown at 1X10^6 cells/mL and analyzed on a fluorescence plate reader or microscope. Plate readers tend to require the most reagent, flow cytometers the least.

How is FLICA™ different from other caspase detection kits?

• The FLICA assay kits are used with whole, living cells; no lysis or permeabilization is necessary.
• FLICA is not an ELISA and does not involve the use of any antibodies. Because active caspase enzymes bind to FLICA, there is no interference from pro-caspases nor inactive forms of the enzyme.
• The fluorescent signal can be analyzed by fluorescence microscopy, plate reader, or flow cytometer.

How soon should the samples be read within labeling?
We recommend reading the cells within 24 hours, as the fluorescent label may photobleach; however, samples have been frozen for 8 weeks and re-analyzed on a plate reader with equivalent results.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.