FAM FLICA™ Caspase 1 Assay Kit

Catalog Number: Select size below to display pricing: 97 (25 tests); 98 (100 tests)

Availability: In stock

$184.00

Quick Overview

Detect caspase-1 activity with the FLICA™ Caspase 1 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe FAM-YVAD-FMK to label active caspase 1 enzyme in living cells or tissue samples. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry.
FLICA (Fluorescent Labeled Inhibitor of Caspases) probes are comprised of an inhibitor peptide sequence that binds to active caspase enzymes, a fluoromethyl ketone (FMK) moiety that results in an irreversible binding event with the enzyme, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. For a caspase 1 inhibitor, the multi-enzyme recognition sequence is tyrosine-valine-alanine-aspartic acid (YVAD). The FLICA™ FAM-YVAD-FMK probe interacts with the enzymatic reactive center of activated caspase 1 via the YVAD recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety.
FLICA™ is cell permeant and non-cytotoxic. Unbound FLICA™ FAM-YVAD-FMK reagent is easily washed away; the remaining green fluorescent signal is a direct measure of caspase 1 activity at the time the probe was added. Detection of nuclear morphology and necrosis is also possible with the additional kit components, Hoechst 33342 and Propidium Iodide, respectively.





Order green FLICA Caspase-1 Assay Kits online or call 800-829-3194.
Catalog no. 97 (25 tests), $184 USD
Catalog no. 98 (100 tests), $504 USD

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Measure Caspase-1 Activity in Whole, Living Cells
Catalog no. 97 (25 tests), $184 USD
Catalog no. 98 (100 tests), $504 USD

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 has also been found to play a role in processing a wide variety of proteins, most notably several cytokines (4-6) and enzymes within the glycolytic pathway (7). The creation of the inflammasome during host responses to pathogens leads to the activation of caspase 1 (8, 9) and may induce the class of cell death known as pyroptosis. 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. Furthermore, 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 probes are in development. To receive pre-release information about ICT's future 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:

Manual for FAM-FLICA Caspase Assay Kits

Reagent Name: FAM-YVAD-FMK

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.
    ABSTRACT
    Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.  PMID: 12126059

  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.
    ABSTRACT
    Activation of caspases is the key event of apoptosis. To detect this event in situ we applied fluorochrome-labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA are fluorescein- or sulforhodamine-conjugated peptide-fluoromethyl ketones that covalently, with 1:1 stoichiometry, bind to enzymatic centers of caspases; the specificity is provided by the peptide sequence of amino acids. Similarly, we applied fluorescent inhibitors of serine proteases (FLISP) to detect active sites of the latter enzymes. Exposure of live cells to FLICA of FLISP led to uptake of these ligands and their binding to activated caspases or active sites of serine proteases; the unbound reagents were removed by cell rinse. Only cells undergoing apoptosis were labeled with FLISP or FLICA. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA or FLISP allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases; two proteins reacting with FLISP were about 57 and 60 kDa, which suggests that they are novel enzymes. Detection of caspases or serine proteases activation can be combined with other markers of apoptosis or cell cycle for multiparametric analysis by flow or laser scanning cytometry. Being caspase inhibitors, FLICA arrest the process of apoptosis and prevent cell disintegration. The stathmo-apoptotic assay was developed, therefore, to obtain cumulative apoptotic index over a long period of time and estimate a rate of cell entry into apoptosis for cell populations. PMID: 12225789

  3. Grunewald, S., Paasch, U., Said, T.M., Sharma, R.K., Glander, H.J., Agarwal, A. 2005. Caspase activation in human spermatozoa in response to physiological and pathological stimuli. Fertil. Steril. 83:1106-1112.
    ABSTRACT
    OBJECTIVE: To investigate caspase activation in response to a variety of pathological and physiological stimuli in light of the fact that current research offers no clear consensus about caspase activation pathways in spermatozoa.
    DESIGN: A prospective, controlled study.
    SETTING: Male infertility clinic, Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio.
    PATIENT(S): Fifteen healthy volunteers.
    INTERVENTION(S): Spermatozoa were exposed to [1] Fibroblast-associated (Fas) death receptor activation, [2] mitochondrial apoptosis induction using betulinic acid, [3] oxidative stress, and [4] prolonged incubation up to 3 hours without any external stimuli.
    MAIN OUTCOME MEASURE(S): Active caspases-1, -3, -8, and -9 were examined in human spermatozoa by flow cytometry using carboxyfluorescein derivatives.
    RESULT(S): Inducing Fas antibody did not result in any caspase activation. Conversely, betulinic acid significantly triggered caspase-9 and -3 activation. The application of oxidative stress and prolonged incubation (3 hours) failed to result in caspase activation.
    CONCLUSION(S): These results suggest that Fas has no functional relevance in mediating caspase activation in human ejaculated spermatozoa. Although spermatozoal mitochondria are highly susceptible to specific agonists of apoptosis such as betulinic acid via caspase activation, oxidative stress-induced apoptosis appears to be caspase independent. PMID: 15831282
  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.
    ABSTRACT
    IL-4 and IL-13 are prototypic Th2 cytokines that generate an "alternatively activated" phenotype in macrophages. We used high-density oligonucleotide microarrays to investigate the transcriptional profile induced in human monocytes by IL-13. After 8-h stimulation with IL-13, 142 genes were regulated (85 increased and 57 decreased). The majority of these genes were related to the inflammatory response and innate immunity; a group of genes related to lipid metabolism was also identified, with clear implications for atherosclerosis. In addition to characteristic markers of alternatively activated macrophages, a number of novel IL-13-regulated genes were seen. These included various pattern recognition receptors, such as CD1b/c/e, TLR1, and C-type lectin superfamily member 6. Several components of the IL-1 system were regulated. IL-1RI, IL-1RII, and IL-1Ra were all up-regulated, whereas the IL-1beta-converting enzyme, caspase 1, and IRAK-M were down-regulated. LPS-inducible caspase 1 enzyme activity was also reduced in IL-13-stimulated monocytes, with a consequent decrease in pro-IL-1beta processing. These data reveal that IL-13 has a potent effect on the transcriptional profile in monocytes. The IL-13-induced modulation of genes related to IL-1 clearly highlights the tightly controlled and complex levels of regulation of the production and response to this potent proinflammatory cytokine. PMID: 15634905
  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.
    ABSTRACT
    The IL-1 family cytokines are regulated on transcriptional and posttranscriptional levels. Pattern recognition and cytokine receptors control pro-IL-1beta transcription whereas inflammasomes regulate the proteolytic processing of pro-IL-1beta. The NLRP3 inflammasome, however, assembles in response to extracellular ATP, pore-forming toxins, or crystals only in the presence of proinflammatory stimuli. How the activation of gene transcription by signaling receptors enables NLRP3 activation remains elusive and controversial. In this study, we show that cell priming through multiple signaling receptors induces NLRP3 expression, which we identified to be a critical checkpoint for NLRP3 activation. Signals provided by NF-kappaB activators are necessary but not sufficient for NLRP3 activation, and a second stimulus such as ATP or crystal-induced damage is required for NLRP3 activation. PMID: 19570822
  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.
    ABSTRACT
    An interesting trait shared by many members of the IL-1 cytokine family is the absence of a signal sequence that can direct the newly synthesized polypeptides to the endoplasmic reticulum. As a result, these cytokines accumulate intracellularly. Recent studies investigating IL-1beta export established that its release is facilitated via activation of an intracellular multiprotein complex termed the inflammasome. The purpose of the current study was to explore the mechanism by which murine IL-1F6 is released from bone marrow-derived macrophages (BMDMs) and to compare this mechanism to that used by IL-1beta. BMDMs were engineered to overexpress IL-1F6 by retroviral transduction; cells overexpressing GFP also were generated to provide a noncytokine comparator. The transduced cells constitutively expressed IL-1F6 and GFP, but they did not constitutively release these polypeptides to the medium. Enhanced release of IL-1F6 was achieved by treating with LPS followed by ATP-induced activation of the P2X(7) receptor; GFP also was released under these conditions. No obvious proteolytic cleavage of IL-1F6 was noted following P2X(7) receptor-induced release. Stimulus-induced release of IL-1F6 and GFP demonstrated comparable susceptibility to pharmacological modulation. Therefore, transduced IL-1F6 is released in parallel with endogenous mature IL-1beta from LPS/ATP-treated BMDMs, but this externalization process is not selective for cytokines as a noncytokine (GFP) shows similar behavior. These findings suggest that IL-1F6 can be externalized via a stimulus-coupled mechanism comparable to that used by IL-1beta, and they provide additional insight into the complex cellular processes controlling posttranslational processing of the IL-1 cytokine family. PMID: 19717513
  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.
    ABSTRACT
    Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K(+) through glibenclamide-sensitive K(+) channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1beta. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1beta, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae. PMID: 19648107
  8. Knodler, et al. 2010. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. PNAS, 107: 17733-17738.
    ABSTRACT
    Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in epithelial cells of the gut and gallbladder. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. Here, we demonstrate that a subpopulation of Salmonella hyperreplicating in the cytosol of epithelial cells serves as a reservoir for dissemination. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen. This extrusion mechanism is morphologically similar to the process of cell shedding required for turnover of the intestinal epithelium. In contrast to the homeostatic mechanism, however, bacterial-induced extrusion is accompanied by an inflammatory cell death characterized by caspase-1 activation and the apical release of IL-18, an important cytokine regulator of gut inflammation. Although epithelial extrusion is obviously beneficial to Salmonella for completion of its life cycle, it also provides a mechanistic explanation for the mucosal inflammation that is triggered during Salmonella infection of the gastrointestinal and biliary tracts. PMID: 20876119
  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.
    ABSTRACT
    Chlamydia trachomatis infections cause severe and irreversible damage that can lead to infertility and blindness in both males and females. Following infection of epithelial cells, Chlamydia induces production of reactive oxygen species (ROS). Unconventionally, Chlamydiae use ROS to their advantage by activating caspase-1, which contributes to chlamydial growth. NLRX1, a member of the Nod-like receptor family that translocates to the mitochondria, can augment ROS production from the mitochondria following Shigella flexneri infections. However, in general, ROS can also be produced by membrane-bound NADPH oxidases. Given the importance of ROS-induced caspase-1 activation in growth of the chlamydial vacuole, we investigated the sources of ROS production in epithelial cells following infection with C. trachomatis. In this study, we provide evidence that basal levels of ROS are generated during chlamydial infection by NADPH oxidase, but ROS levels, regardless of their source, are enhanced by an NLRX1 dependent mechanism. Significantly, the presence of NLRX1 is required for optimal chlamydial growth. PMID: 20959452
  10. Broz, P., Newton, K., Lamkanfi, M., Mariathasan, S., Dixit, V. M., & Monack, D. M. (2010). Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against SalmonellaJ exp med207(8), 1745-1755.
    EXCERPT: "... FAM-YVAD-FMK stainings (FLICA; ImmunoChemistry Technologies), the medium was removed 1 h before the collection time point and replaced with fresh DME containing 5 µM FLICA. For caspase-1 inhibitor treatments, the macrophages were cultured in 20 µM Z-YVAD ..."
  11. Broz, P., von Moltke, J., Jones, J. W., Vance, R. E., & Monack, D. M. 2010. Differential Requirement for Caspase-1 Autoproteolysis in Pathogen-Induced Cell Death and Cytokine ProcessingCell host & microbe8 (6), 471-483.
    ABSTRACT
    Activation of the cysteine protease Caspase-1 is a key event in the innate immune response to infections. Synthesized as a proprotein, Caspase-1 undergoes autoproteolysis within multiprotein complexes called inflammasomes. Activated Caspase-1 is required for proteolytic processing and for release of the cytokines interleukin-1β and interleukin-18, and it can also cause rapid macrophage cell death. We show that macrophage cell death and cytokine maturation in response to infection with diverse bacterial pathogens can be separated genetically and that two distinct inflammasome complexes mediate these events. Inflammasomes containing the signaling adaptor Asc form a single large “focus” in which Caspase-1 undergoes autoproteolysis and processes IL-1β/IL-18. In contrast, Asc-independent inflammasomes activate Caspase-1 without autoproteolysis and do not form any large structures in the cytosol. Caspase-1 mutants unable to undergo autoproteolysis promoted rapid cell death, but processed IL-1β/18 inefficiently. Our results suggest the formation of spatially and functionally distinct inflammasomes complexes in response to bacterial pathogens.
  12. Liu, G, Fiala, M, Mizwicki, MT, Sayre, J, Magpantay, L, Siani, A, Mahanian, M, Chattopadhyay, M, La Cava, A, and M Wiedau-Pazos. 2012. Neuronal phagocytosis by inflammatory macrophages in ALS spinal cord: inhibition of inflammation by resolvin D1Am J Neurodegener Dis 2012;1(1):60-74.
    ABSTRACT
    Although the cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS) remains hypothetical, there is evidence of spinal cord infiltration by macrophages and T cells. In post-mortem ALS spinal cords, 19.8 + 4.8 % motor neurons, including caspase–negative and caspase-positive neurons, were ingested by IL-6- and TNF-α-positive macrophages. In ALS macrophages, in vitro aggregated superoxide dismutase-1 (SOD-1) stimulated in ALS macrophages expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, through activation of cyclooxygenase-2 (COX-2) and caspase-1. The lipid mediator resolvin D1 (RvD1) inhibited IL-6 and TNF-α production in ALS macrophages with 1,100 times greater potency than its parent molecule docosahexaenoic acid. ALS peripheral blood mononuclear cells (PBMCs) showed increased transcription of inflammatory cytokines and chemokines at baseline and after stimulation by aggregated wild-type SOD-1, and these cytokines were down regulated by RvD1. Thus the neurons are impacted by macrophages expressing inflammatory cytokines. RvD1 strongly inhibits in macrophages and PBMCs cytokine transcription but does not inhibit their production in PBMCs. Resolvins offer a new approach to ALS inflammation suppressing. (AJND1103001)
  13. Xiong, J., & Kielian, T. 2013. Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotypeJ neurochem127(2), 245-258.
    ABSTRACT
    Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro-IL-1β in microglia, and IL-1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3Δex7/8mutant and wild-type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL-1β release in CLN3Δex7/8 microglia but not WT cells, as well as increased expression of additional pro-inflammatory mediators. Similar effects were observed following either TNF-α or IL-1β treatment, suggesting that CLN3Δex7/8 microglia exist in primed state and hyper-respond to several inflammatory stimuli compared to WT cells. CLN3Δex7/8 microglia displayed constitutive caspase-1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3Δex7/8 or WT microglia induced significant cell death in CLN3Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3Δex7/8 neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.
    Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. In this report, primary microglia from CLN3Δex7/8 mutant mice over-produced numerous inflammatory cytokines in response to stimuli that are present in the JNCL brain, whereas wild-type microglia were relatively non-responsive. In addition, activated microglia induced significant cell death in CLN3Δex7/8 but not wild-type neurons, demonstrating that intrinsically diseased CLN3Δex7/8neurons are less equipped to withstand cytotoxic insults. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.
    Keywords: caspase-1; CLN3; IL-1β; inflammasome; juvenile Batten disease;microglia
    EXCERPT:
    "... that astrocytes do not produce pro-inflammatory cytokines (ie IL-1β or TNF-α) (Liu and Kielian 2011; Holm et ... Caspase-1 activation was analyzed using the FLICA reagent (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's instructions and ..."
  14. Kielian, Tammy. "Compositions and methods for the treatment of juvenile neuronal ceroid lipofuscinosis and related disorders." U.S. Patent Application 14/066,506, filed October 29, 2013.
    EXCERPT:
    "... Inventors, Tammy Kielian... day 2) were treated with 100 ng/ml LPS+5 mM ATP; 10 μg/ml C6 ceramide, and primary neuronal lysates either alone or in combination for 6 hours, whereupon caspase-1 activation was quantitated by FACS using the FLICA reagent (Immunochemistry ...

Caspase 1 data

 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).

 

 

 

 

 

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Target: caspase 1
Excitation / Emission: 488 nm / 530 nm
Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
Types of Samples: cell culture, tissue
Kit Contents: Kit #97 (Small Size, 25 tests):
  • FLICA Caspase 1 Reagent (FAM-YVAD-FMK), 1 vial
  • 10x Apoptosis Wash Buffer, 15mL
  • Fixative, 6mL
  • Hoechst 33342 Stain, 1mL
  • Propidium Iodide Stain, 1mL

Kit #98 (Large size, 100 tests):

  • FLICA Caspase 1 Reagent (FAM-YVAD-FMK), 4 vials
  • 10x Apoptosis Wash Buffer, 60mL
  • Fixative, 6mL
  • Hoechst 33342 Stain, 1mL
  • Propidium Iodide Stain, 1mL

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

Kit Component MSDS

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 30 mL of cell culture samples - approximately 100 tests. The number of tests to be achieved per FLICA vial depends on sample size, application, and preferred working concentration.

What is one "test"?
For the sake of a general recommendation, one "test" is a 300 uL aliquot of cells grown to 2-6 X 106 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 the FLICA™ method different from other caspase detection assays?

  • Cell permeant reagents enable imaging and cytometry applications.
  • 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 or substrates. Active caspase-1 enzymes will bind to FLICA 660-YVAD-FMK within the living cell, so there will be no interference from pro-caspases or lysing procedures.
  • Flexible multiplexing is possible with the use of additional fluorescent dyes or probes.

How soon should the samples be read within labeling?
If cells are not to be read immediately after the staining and wash steps, we recommend fixing or freezing the samples and reading the cells within 16 hours. Protect from light as the fluorescent label may photobleach. However, samples have been frozen for 8 weeks and re-analyzed on a plate reader or microscope with equivalent results.

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