FLICA® 660 Caspase-1 Assay Kit, far-red fluorescence

Catalog Number: 9122 (25-50 tests)

Availability: In stock

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Quick Overview

Forget the lysates. Detect caspase-1 activation in whole, living cells with the FLICA® 660 Caspase-1 Assay Kit. This in vitro caspase-1 assay employs a new far-red fluorescent caspase-1 inhibitor detection probe, FLICA® 660-YVAD-FMK, to label activated caspase-1 enzyme in living cells or tissue samples. Image the fluorescent signal using fluorescence microscopy or analyze samples for caspase-1 activation by flow cytometry.

  • Ex: 660 nm / Em: 680-690 nm

  • Whole cell analysis via fluorescence microscopy or flow cytometry

  • Flexible multiplexing with additional dyes or probes

  • Compatibility with GFP and green autofluorescence

  • Benchtop flow compatible

The cell permeant probe in the FLICA caspase-1 assay, 660-YVAD-FMK, is comprised of an affinity inhibitor peptide sequence (YVAD) and a fluoromethyl ketone (FMK) moiety that enable an irreversible, covalent binding event with activated caspase-1 enzymes in whole, living cells. The new FLICA probe is labeled with a far-red fluorescent 660 dye reporter, enabling detection via common fluorescence detection methods.

Order FLICA 660 Caspase-1 Assay Kit online or call 800-829-3194.
Catalog no. 9122 (25-50 tests)

FLICA® 660 Caspase-1 Assay Kit, far-red fluorescence

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  • THP-1 cells with activated caspase-1 (ICE) emit far-red fluorescence after staining with FLICA 660-YVAD-FMK

Detect Caspase-1 Activation in Whole, Living Cells
FLICA® 660 Caspase-1 Assay Kit, Catalog no. 9122

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 -1 beta (IL-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 the pro-inflammatory cytokine Interleukin 1 beta (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) and the class of cell death known as pyroptosis. 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® Method to Detect Caspase-1 Activation
The FLICA® 660 Caspase-1 Assay utilizes the red caspase-1 detection probe, 660-YVAD-FMK, which is comprised of the preferred affinity peptide sequence for active caspase-1 (YVAD), a far-red fluorescent 660 dye label, and a fluoromethyl ketone (FMK) reactive moiety. The resulting fluorescent caspase-1 inhibitor probe forms an irreversible, covalent bond with the active caspase-1 enzyme, efficiently labeling the target for detection. Due to its cell permeant nature and fluorescence properties, the red FLICA caspase-1 probe enables whole cell analysis via common fluorescence detection methods.

To use FLICA, add it directly to suspension cell or tissue culture media, incubate, and wash. The cell permeant FLICA 660-YVAD-FMK caspase-1 detection reagent will efficiently diffuse into cells and irreversibly bind to activated caspase-1 enzymes, thereby retaining the red signal inside caspase-1-positive cells. Cells not bearing active caspase-1 return to a non-fluorescent status after the wash step. The FLICA® 660 Caspase-1 probe has an optimal excitation at 660 nm and optimal emission range from 680-690 nm. As such, it has demonstrated excellent excitation efficiency with a conventional red HeNe laser with a 633 nm excitation, enabling samples to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. Cells labeled with the FLICA® reagent may be read immediately or preserved for 16 hours using the fixative.

Recommendations for use:
FLICA 660-YVAD-FMK caspase-1 detection probe is supplied as stable, lyophilized reagent, which is reconstituted with DMSO and diluted to working solution with PBS just prior to use. Each vial provides 250 uL of the FLICA 660-YVAD-FMK working solution that is to be used immediately after preparation.

FLICA 660-YVAD-FMK working solution is added to suspension cell samples at a 1:30- 1:60 ratio. This calculates to 5-10 uL of working solution used per 300 uL cell sample. The optimal ratio depends on the application and analysis method. Flow cytometry analysis provides the sensitivity to detect FLICA 660-YVAD-FMK when used at 1:60. Microscopy analysis may require a higher staining concentration of FLICA 660-YVAD-FMK than flow cytometry, so we recommend using the FLICA working solution at 1:30 for this type of analysis.

Additional FLICA caspase probes are in development. To receive pre-release information about ICT's new caspase 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.
Product Manuals:

Download Manual for FLICA 660 Caspase-1 Assay Kit

Reagent Name: 660-YVAD-FMK

Sample Protocol:

FLICA™ (Fluorescent-Labeled Inhibitor of Caspases) assays offer a simple yet accurate method to measure individual or pan-caspase activity in whole cells. Two sample protocols are outlined below. Detailed instructions are supplied with the kit.

Suspension Cells

  1. Culture your cells to a concentration of 2-5 x 105 cells/mL.
  2. Prepare experimental and control populations.
  3. Reconstitute the reagent with 50µL DMSO to form the stock concentrate (may be frozen for future use).
  4. Dilute the stock concentrate with 200µL PBS to form the working solution.
  5. Add working solution directly to samples and controls at a ratio of 1:30 – 1:60. E.g., add 5-10 µL of working solution to a 300 uL aliquot of suspension cells. Flow cytometry requires less reagent; microscopy applications require more reagent per sample.
  6. Incubate 15 - 45 minutes. 
  7. Wash and spin cells two or three times.
  8. If desired, label cells with Hoechst stain (cat. #639), DAPI (cat. #6244), or other compatible fluorescent markers.
  9. If desired, fix cells with fixative included in kits.
  10. Analyze data using a fluorescence microscope or flow cytometer.

Tissue Sections

  1. Prepare experimental and control populations.
  2. Prepare thin frozen tissue sections appropriate to the experiment. Allow sections to thaw before staining with FLICA reagent.
  3. Reconstitute each vial of FLICA with 50 uL DMSO to form the stock concentrate.
  4. Dilute FLICA stock concentrate 1:50 in PBS to form the tissue section staining solution (TSSS). Use TSSS within 15 minutes of preparation. 
  5. Add enough TSSS to cover the surface of the tissues and incubate 30 - 60 minutes protected from light. 
  6. Wash with TBSt, PBSt, or 1X Cellular Wash Buffer (twice for 5 min).
  7. Set slides in slide incubation dish containing 1X Cellular Wash Buffer.
  8. Stain nuclei with DAPI (cat. #6244) or Hoechst (cat. #639), and apply coverslip.
  9. Image with appropriate fluorescence microscopy filters.
  10. Store samples at 2-8°C for short-term storage; staining will last at -20° C for long periods.

  1. Miles, B., Scisci, E., Carrion, J., Sabino, G. J., Genco, C. A., & Cutler, C. W. (August 2013). Noncanonical dendritic cell differentiation and survival driven by a bacteremic pathogenJ leukocyte biol94(2), 281-289.
    "... Intracellular polycaspase, initiator caspase-3/7, and effector caspase-1 and -8 were determined using Vybrant FAM FLICA kits (V3511-7, -8, -9; Molecular Probes, Life Technologies) and FLICA 660 Caspase-1 kit (9122; ImmunoChemistry Technologies, Bloomington, MN, USA ..."

    "Maintenance of blood DC homeostasis is essential to preventing autoimmunity while controlling chronic infection. However, the ability of bacteremic pathogens to directly regulate blood DC homeostasis has not been defined. One such bacteremic pathogen, Porphyromonas gingivalis, is shown by our group to survive within mDCs under aerobic conditions and therein, metastasize from its oral mucosal niche. This is accompanied by expansion of the blood mDC pool in vivo, independently of canonical DC poietins. We presently know little of how this bacteremic pathogen causes blood DC expansion and the pathophysiological significance. This work shows that optimum differentiation of MoDCs from primary human monocytes, with or without GM-CSF/IL-4, is dependent on infection with P. gingivalis strains expressing the DC-SIGN ligand mfa-1. DC differentiation is lost when DC-SIGN is blocked with its ligand HIV gp120 or knocked out by siRNA gene silencing. Thus, we have identified a novel, noncanonical pathway of DC differentiation. We term these PDDCs and show that PDDCs are bona fide DCs, based on phenotype and phagocytic activity when immature and the ability to up-regulate accessory molecules and stimulate allo-CD4+ T cell proliferation when matured. The latter is dependent on the P. gingivalis strain used to initially “educate” PDDCs. Moreover, we show that P. gingivalis-infected, conventional MoDCs become resistant to apoptosis and inflammatory pyroptosis, as determined by levels of Annexin V and caspase-8, -3/7, and -1. Taken together, we provide new insights into how a relatively asymptomatic bacteremia may influence immune homeostasis and promote chronic inflammation."
    KEYWORDS: inflammation, apoptosis, porphyromonas gingivalis, periodontal pathogen, T cells

  2. Mortimer, L., Moreau, F., Cornick, S., & Chadee, K. (November 2013). Gal-lectin-dependent contact activates the inflammasome by invasive Entamoeba histolytica. Mucosal immunology. doi:10.1038/mi.2013.100.
    "... in cold acetone for 5 min. YVAD-FLICA (FLICA 660-Caspase-1 reagent, Immunochemistry Technologies, Bloomington, MN) at 1:70 dilution was added for 1 h at room temperature. Cells were washed twice in phosphate-buffered ..."

    "Entamoeba histolytica (Eh) is an extracellular protozoan parasite of the human colon, which occasionally breaches the intestinal barrier. Eradicating ameba that invades is essential for host survival. A defining but uncharacterized feature of amebic invasion is direct contact between ameba and host cells. This event corresponds with a massive pro-inflammatory response. To date, pathogen recognition receptors (PRRs) that are activated by contact with viable Eh are unknown. Here we show that the innate immune system responds in a qualitatively different way to contact with viable Eh vs. soluble ligands produced by viable or dead ameba. This unique Eh Gal-lectin contact-dependent response in macrophages was mediated by activation of the inflammasome. Soluble native Gal-lectin did not induce inflammasome activation, but was sufficient for transcriptional priming of the inflammasome and non-inflammasome-dependent pro-inflammatory cytokine release. We conclude the inflammasome is a pathogenicity sensor for invasive Eh and identify for the first time a PRR that specifically responds to contact with intact parasites in a manner that accords with scale immune response to parasite invasion."
  3. Doitsh, Gilad, et al. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infectionNature 505.7484 (2014): 509-514.
    "... Caspase 1 activity was determined by flow cytometry using FLICA 12 h after treatment with nigericin (10 μM) or 3 days after infection with HIV. ... 19), indicating a specific recognition of caspase 1 and caspase 3 activity by the FLICA probes. ..."

    "The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection—CD4 T-cell depletion and chronic inflammation—and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of ‘anti-AIDS’ therapeutics targeting the host rather than the virus."
  4. Wree, Alexander, Akiko Eguchi, Matthew D. McGeough, Carla A. Pena, Casey D. Johnson, Ali Canbay, Hal M. Hoffman, and Ariel E. Feldstein. (2014). NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice. Hepatology59(3), 898-910.
    "... Active Casp1 was measured in parenchymal cell suspensions with FLICA 660-YVAD-FMK (FLICA 660 in vitro Active Caspase-1 Detection Kit; ImmunoChemistry Technologie {sic}..."

    "Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry–based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Conclusions: Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage. (Hepatology 2013;)"
  5. Monroe, K. M., Yang, Z., Johnson, J. R., Geng, X., Doitsh, G., Krogan, N. J., & Greene, W. C. (2014). IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIVScience 343(6169), 428-432.
    "... HIV-1 D116N integrase mutant. **P < 0.01, *P < 0.05. (F) Flow cytometric analysis of FLICA-660 caspase-1 and IFN-β intracellular staining in mCherry + cells. Histograms are representative of results obtained from two donors. ..."

    "The progressive depletion of quiescent “bystander” CD4 T cells, which are nonpermissive to HIV infection, is a principal driver of the acquired immunodeficiency syndrome (AIDS). These cells undergo abortive infection characterized by the cytosolic accumulation of incomplete HIV reverse transcripts. These viral DNAs are sensed by an unidentified host sensor that triggers an innate immune response, leading to caspase-1 activation and pyroptosis. Using unbiased proteomic and targeted biochemical approaches, as well as two independent methods of lentiviral short hairpin RNA–mediated gene knockdown in primary CD4 T cells, we identify interferon-γ–inducible protein 16 (IFI16) as a host DNA sensor required for CD4 T cell death due to abortive HIV infection. These findings provide insights into a key host pathway that plays a central role in CD4 T cell depletion during disease progression to AIDS."

We expect to see more citations of the far-red fluorescent FLICA® 660 Caspase-1 Assay Kit soon. The FLICA 660-YVAD-FMK probe in this new caspase-1 assay is a far-red analog to the green fluorescent caspase-1 detection probe, FLICA® FAM-YVAD-FMK, which is cited regularly. The following is a list of selected literature citations for Green FLICA® Caspase-1 Assay Kit (FAM-YVAD-FMK).

  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, TM, Sharma, RK, Glander, HJ, Agarwal, A. 2005. Caspase activity in human spermatozoa in response to physiological and pathological stimuli. Fertil Steril, 83:1106-1112.
  4. Scotton, CJ, Martinez, FO, Smelt, MJ, 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. Andrew J. Grant, Mark Sheppard, Rob Deardon, Sam P. Brown, Gemma Foster, Clare E. Bryant, Duncan J. Maskell and Pietro Mastroeni. 2008. Caspase-3-dependent phagocyte death during systemic Salmonella enterica serovar Typhimurium infection of mice. J Immunol
  6. 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.
  7. 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.
  8. 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.
  9. Knodler, L. et al. 2010. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. PNAS, 107: 17733-17738.
  10. 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.
  11. Petr Broz, Jakob von Moltke, Jonathan W. Jones, Russell E. Vance, and Denise M. Monack. 2010. Differential Requirement for Caspase-1 Autoproteolysis in Pathogen-Induced Cell Death and Cytokine Processing. Cell Host Microbe
  12. Felix Meissner, Reinhard A. Seger, Despina Moshous, Alain Fischer, Janine Reichenbach, and Arturo Zychlinsky. 2011. Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease. Blood
  13. Murphey, ED. 2011. Cecal Ligation and Puncture-Induced Impairment of Innate Immune Function Does Not Occur in the Absence of Caspase-1. J Immunol, 187: 905 - 910.
  14. Mélanie Anne Hamon and Pascale Cossart. 2011. K+ Efflux Is Required for Histone H3 Dephosphorylation by Listeria monocytogenes Listeriolysin O and Other Pore-Forming Toxins. Infect Immun, 79: 2839 - 2846.
  15. Norma Olivares-Zavaleta, Aaron Carmody, Ronald Messer, William M. Whitmire, and Harlan D. Caldwell. 2011. Chlamydia pneumoniae Inhibits Activated Human T Lymphocyte Proliferation by the Induction of Apoptotic and Pyroptotic Pathways. J Immunl, 186:7120-7126.
  16. Maninjay K. Atianand and Jonathan A. Harton. 2011. Uncoupling of Pyrin-only Protein 2 (POP2)-mediated Dual Regulation of NF-B and the Inflammasome. J Biol Chem, 286: 40536 - 40547.

Excitation Spectrum of FLICA 660 Reagents Emission Spectrum of FLICA 660 Reagents


Excitation max: 660 nm
Emission max: 690 nm



THP-1 cells induced for Caspase-1 activity are detected by flow cytometry and 660-YVAD-FMK inhibitor probe



THP-1 cells were treated with either a placebo or PMA and LPS. After treatment and trypsinization, FLICA 660-YVAD-FMK far-red caspase-1 reagent was added to the culture media.

Flow cytometric analysis of the positive control sample (top) reveals a slight increase in cells bearing active caspase-1 enzyme when compared to the population of placebo-treated cells (bottom). An 8:1 differential was achieved with the positive control treatment.




Negative control sample of non-differentiated THP-1 cells analyzed for caspase-1 activity by flow cytometry and 660-YVAD-FMK FLICA inhibitor probe







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!

Target: caspase 1
Excitation / Emission: 660 nm / 690 nm
Method of Analysis: Flow Cytometer, Fluorescence Microscope
Types of Samples: cell culture, tissue
Kit Contents: Kit #9122 (25-50 tests)
  • FLICA 660 Caspase-1 Reagent (660-YVAD-FMK), 1 vial
  • 10X Cellular Wash Buffer, 15 mL
  • Fixative, 6 mL
  • Technical Datasheet and Protocol



Storage: 2°-8° C, Ships Overnight (Domestic), International Priority Shipping

FLICA® 660 Caspase-1 Assay Kit MSDS

How many tests can be run with the assay kit?
The FLICA 660 Caspase-1 Assay Kit (#9122) provides enough reagent to test 7.5 - 15mL of cell culture samples - approximately 25-50 tests.

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

What are your recommendations for use? 
FLICA® 660-YVAD-FMK working solution is added to suspension cell samples at a 1:30- 1:60 ratio. The optimal ratio depends on the application and analysis method. Flow cytometry analysis provides the sensitivity to detect FLICA 660-YVAD-FMK when used at 1:60. Microscopy analysis may require a higher staining concentration of FLICA 660-YVAD-FMK than flow cytometry, so we recommend using the FLICA working solution at 1:30 for this type of analysis. We recommend 300 µL sample sizes, aliquoted from cell cultures grown to a concentration of 2-5 x 105 cells/mL. In this manner, 25-50 tests per vial may be achieved. Different sample volumes may be used, which changes the amount of FLICA to be used per sample and alters the number of tests per vial accordingly.

Recommendations for Flow Cytometry: For analysis by flow cytometry, sufficient staining may be achieved by using the FLICA 660-YVAD-FMK working solution at a 1:60 ratio in suspension cells at 2-5 x 105 cells/mL. E.g., add 5 µL of working solution to a 300 µL aliquot of cells. In this particular example, 50 tests per vial of FLICA 660-YVAD-FMK are achieved; actual number of tests will increase or decrease depending on sample size.
With its optimal emission peak at 680-690 nm, the signal from far-red FLICA 660-YVAD-FMK can be easily read in the FL-4 channel.

Recommendations for Fluorescence Microscopy: This application requires a higher concentration of cells per mL and a higher staining concentration of FLICA® 660-YVAD-FMK reagent when compared with flow cytometric analysis methods. To provide an adequate cell density within the field of vision at higher magnifications, the cells should be concentrated by centrifugation to 2-5 x 106 cells/mL just prior to staining with FLICA 660-YVAD-FMK. (The initial cell concentration in cultures should be between 2 – 5 x 105 cells/mL.) 
Accordingly, analysis by fluorescence microscopy also requires a higher staining concentration of FLICA 660-YVAD-FMK reagent. For this analysis method, use the FLICA 660-YVAD-FMK working solution at a 1:30 ratio in the cell suspension, e.g., add 10 µL of working solution to a 300 µL aliquot of cells. In this particular example, 25 tests per vial of FLICA 660-YVAD-FMK are achieved; actual number of tests will increase or decrease depending on sample size.

In the protocol, a large kit #9123 is mentioned, but it is not on your website. Is a larger size available?
A larger size of the FLICA 660 Caspase-1 Assay Kit is currently not offered. The manual for kit #9122 was intended for use with both sizes when another size is launched.

We are collecting customer feedback on this relatively new assay before introducing a larger size. If you would like to provide feedback on this assay's packaging options (size, content, etc), please contact us via our webform or call the ICT Product Development team at 1-800-829-3194.

With our various flow cytometry controls, we might need more Cellular Wash Buffer. May we acquire additional Cellular Wash Buffer?
The 15 mL bottle of 10X Cellular Wash Buffer in kit #9122 should be sufficient to wash 75 cell aliquots (controls from Section 7.3 of manual and experimental test samples). If additional buffer is required, please request a 15 mL (#6164) or a 60 mL (#6165) bottle of 10X Cellular Wash Buffer on your order form. In addition to 1X Cellular Wash Buffer, cell culture media containing FBS or other additives may be used to wash cells.

How is FLICA™ different from other caspase assays and 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.