Caspase-8 (active) FITC Staining Kit (ab65614)

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Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)
  • Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)
  • Flow Cytometry - Caspase 8 (active) FITC Staining Kit (ab65614)

Key features and details

  • Assay type: Enzyme activity
  • Platform: Microplate reader, Fluor. microscope, Flow cyt.
  • Assay time: 2 hr
  • Sample type: Adherent cells, Suspension cells

Overview

  • Product name

    Caspase-8 (active) FITC Staining Kit
    See all Caspase-8 kits

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Enzyme activity

  • Assay time

    2h 00m

  • Product overview

    Caspase 8 (active) FITC Staining Kit (ab65614) provides a convenient means for sensitive detection of activated caspase 8 in living cells. The assay utilizes the caspase 8 inhibitor, IETD-FMK, conjugated to FITC (FITC-IETD-FMK) as a marker. FITC-IETD-FMK is cell permeable, non-toxic, and irreversibly binds to activated caspase 8 in apoptotic cells. The FITC label allows detection of activated caspase-8 in apoptotic cells directly by fluorescence microscopy, flow cytometry, or fluorescence plate reader.
    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Activation of caspases plays a central role in apoptosis.

    Other caspase and apoptosis assays

    Review the full set of caspase assays, or the apoptosis assay and apoptosis marker guide.

  • Platform

    Microplate reader, Fluor. microscope, Flow cyt.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    FITC-IETD-FMK 1 x 100µl
    Wash Buffer 2 x 100ml
    Z-VAD-FMK 1 x 10µl

  • Research areas

  • Function

    Most upstream protease of the activation cascade of caspases responsible for the TNFRSF6/FAS mediated and TNFRSF1A induced cell death. Binding to the adapter molecule FADD recruits it to either receptor. The resulting aggregate called death-inducing signaling complex (DISC) performs CASP8 proteolytic activation. The active dimeric enzyme is then liberated from the DISC and free to activate downstream apoptotic proteases. Proteolytic fragments of the N-terminal propeptide (termed CAP3, CAP5 and CAP6) are likely retained in the DISC. Cleaves and activates CASP3, CASP4, CASP6, CASP7, CASP9 and CASP10. May participate in the GZMB apoptotic pathways. Cleaves ADPRT. Hydrolyzes the small-molecule substrate, Ac-Asp-Glu-Val-Asp-
    -AMC. Likely target for the cowpox virus CRMA death inhibitory protein. Isoform 5, isoform 6, isoform 7 and isoform 8 lack the catalytic site and may interfere with the pro-apoptotic activity of the complex.

  • Tissue specificity

    Isoform 1, isoform 5 and isoform 7 are expressed in a wide variety of tissues. Highest expression in peripheral blood leukocytes, spleen, thymus and liver. Barely detectable in brain, testis and skeletal muscle.

  • Involvement in disease

    Defects in CASP8 are the cause of caspase-8 deficiency (CASP8D) [MIM:607271]. CASP8D is a disorder resembling autoimmune lymphoproliferative syndrome (ALPS). It is characterized by lymphadenopathy, splenomegaly, and defective CD95-induced apoptosis of peripheral blood lymphocytes (PBLs). It leads to defects in activation of T-lymphocytes, B-lymphocytes, and natural killer cells leading to immunodeficiency characterized by recurrent sinopulmonary and herpes simplex virus infections and poor responses to immunization.

  • Sequence similarities

    Belongs to the peptidase C14A family.
    Contains 2 DED (death effector) domains.

  • Domain

    Isoform 9 contains a N-terminal extension that is required for interaction with the BCAP31 complex.

  • Post-translational
    modifications

    Generation of the subunits requires association with the death-inducing signaling complex (DISC), whereas additional processing is likely due to the autocatalytic activity of the activated protease. GZMB and CASP10 can be involved in these processing events.
    Phosphorylated upon DNA damage, probably by ATM or ATR.

  • Cellular localization

    Cytoplasm.
  • Information by UniProt

  • Alternative names

    • ALPS2B
    • Amyotrophic lateral sclerosis 2 chromosomal region candidate gene 12 protein
    • Apoptosis related cysteine peptidase
    • Apoptotic cysteine protease
    • Apoptotic protease Mch-5
    • Apoptotic protease Mch5
    • CAP 4
    • CAP4
    • CASP-8
    • CASP8
    • CASP8_HUMAN
    • Caspase 8
    • Caspase 8 apoptosis related cysteine peptidase
    • Caspase IIX
    • Caspase-8 subunit p10
    • caspase8
    • CED 3
    • FADD Homologous ICE/CED3 Like Protease
    • FADD Like ICE
    • FADD-homologous ICE/CED-3-like protease
    • FADD-like ICE
    • FLICE
    • FLJ17672
    • ICE-like apoptotic protease 5
    • MACH
    • MACH alpha 1/2/3 protein
    • MACH beta 1/2/3/4 protein
    • MACH5
    • MCH 5
    • MCH5
    • MGC78473
    • MORT1 associated ced 3 homolog
    • MORT1 associated CED3 homolog
    • MORT1-associated CED-3 homolog
    • OTTHUMP00000163717
    • OTTHUMP00000163720
    • OTTHUMP00000163724
    • OTTHUMP00000163725
    • OTTHUMP00000165062
    • OTTHUMP00000165063
    • OTTHUMP00000165064
    • OTTHUMP00000206552
    • OTTHUMP00000206582
    see all

Images

  • Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)
    Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)

    Active caspase 8 in control Jurkat cells (10e6/mL) or cells treated for five hours with 10 ug/mL Cyclohexamide (CHX) (ab120093) or four hours with 25 ug/mL Mitomycin C (MitoC) (ab120797). Background signal subtracted, duplicates; +/- SD.

  • Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)
    Functional assays: Caspase 8 (active) FITC Staining Kit (ab65614)

    Active caspase 8 in Jurkat cells following four hours exposure to 50 ng/mL anti-Fas Ab (αFas) (MBL), five hours with 10 μg/mL cyclohexamide (CHX) (ab120093), or one hour pretreatment with CHX followed by four hours with αFas. Background signal subtracted, duplicates; +/- SD.

  • Flow Cytometry - Caspase 8 (active) FITC Staining Kit (ab65614)
    Flow Cytometry - Caspase 8 (active) FITC Staining Kit (ab65614)Hernandez-Flores G et al., BMC Cancer, 11, 483, 2011 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/2.0
    Caspase 8 activation of Hela (left) and SiHa (right) cells after in vitro treatment with pentoxylline (PTX) or cisplatin (CIS) either alone or in combination. Results represent the mean ± SD of three independent experiments carried out in triplicate. (*) p<0.001 vs CTL.

    Image obtained from Hernandez-Flores G et al; BMC Cancer, 2011 Nov 11; 11:483

References (1)

Publishing research using ab65614? Please let us know so that we can cite the reference in this datasheet.

ab65614 has been referenced in 1 publication.

  • Hernandez-Flores G  et al. Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence. BMC Cancer 11:483 (2011). PubMed: 22074157

Customer reviews and Q&As

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Question

1.) For quantification of staining by Flow Cytometry, what should I use as an isotype control? 2.) I would like to analyze live cells that are undergoing apoptosis, can you please suggest a viability dye that can be used with this kit? 3.) In step 1 of the staining procedure, you recommend preparing a negative control using Z-VAD-FMK to inhibit caspase activation. How long should I incubate my cells with this inhibitor?

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Answer

Thank you for contacting us. 1.) You may use Caspase-8 negative cells (cells that were not stimulated to induce Caspase-8) for your negative control in Flow Cytometry. 2.) For a viability dye, you may try using PI but this will need to be optimized for your applications. 3.) The Z-VAD-FMK inhibitor may be incubated for 30-60 minutes or for the same amount of time that your test inhibitor is incubated with the cells. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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