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FAM-FLICA® Caspase-8 Assay Kit


Detect caspase-8 activity with the FLICA Caspase-8 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe FAM-LETD-FMK to label active caspase-8 enzyme in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

Catalog # Size Quantity Price
99 25 Tests $195.00
910 100 Tests $561.00
Catalog #: 99, 910 Categories: , , ,

Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells and tissues. The FAM FLICA Caspase-8 assay probe allows researchers to assess caspase-8 activation.

The FLICA reagent FAM-LETD-FMK enters each cell and irreversibly binds to activated caspase-8. Because the FAM-LETD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound FAM-LETD-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-8 enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by a fluorescence plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative included in the kit. Unfixed samples may also be analyzed with Propidium Iodide or Hoechst 33342 to detect necrosis or changes in nuclear morphology, respectively.

1. Prepare samples and controls
2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
3. Reconstitute FLICA with 50 μL DMSO.
4. Dilute FLICA 1:5 by adding 200 μL PBS.
5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 μL to 290 μL of cultured cells).
6. Incubate approximately 1 hour.
7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.
8. If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.
9. If desired, fix cells.
10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excites at 492 nm and emits at 520 nm.

If working with adherent cells, please see the manual for additional protocols.

ICT’s Caspase-8 FLICA kit was used to detect apoptosis in U937 cells, a histiocytic lymphoma cell line. Cells were treated with a control, 50 ng/mL TNF, or 50 µg/mL cycloheximide (CHX) for 24 hours. Cells were then incubated with FAM-LETD-FMK for 1 hour, washed 4 times, then analyzed on a flow cytometer. FLICA revealed that 41.9% of cells treated with CHX had caspase-8 activity, compared with 19.1% of cells treated with TNF, and only 1.9% of the control cells (Ms. Jennifer Mitchell, Scripps).

• Reagent name: FAM-LETD-FMK
• Target: Caspase-8
• Excitation/Emission: 492 nm / 520 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 99: 25 Tests
• FLICA Caspase-8 Reagent (FAM-LETD-FMK), 1 vial, #656
• 10X Apoptosis Wash Buffer, 15 mL, #635
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 910: 100 Tests
• FLICA Caspase-8 Reagent (FAM-LETD-FMK), 4 vials, #656
• 10X Apoptosis Wash Buffer, 60 mL, #635
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Lima, TS;Mallya, S;Jankeel, A;Messaoudi, I;Lodoen, MB. Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases. mBio. 2021 Jan 26; 12(1). doi: 10.1128/mBio.02031-20. Full Article

Suzuki T, Kohyama K, Moriyama K, Ozaki M, Hasegawa S, Ueno T, Saitoe M, Morio T, Hayashi M, Sakuma H. Extracellular ADP augments microglial inflammasome and NF-κB activation via the P2Y12 receptor. Eur J Immunol. 2019 Sep 24. doi: 10.1002/eji.201848013. [Epub ahead of print]. https://onlinelibrary.wiley.com/doi/abs/10.1002/eji.201848013. Abstract

Gornowicz A, Pawłowska N, Czajkowska A, Czarnomysy R, Bielawska A, Bielawski K, Michalak O, Staszewska-Krajewska O, and Kałuża Z. Biological evaluation of octahydropyrazin[2,1-a:5,4-a’]diisoquinoline derivatives as potent anticancer agents. Tumour Biol. 2017. Jun;39(6):1010428317701641. doi: 10.1177/1010428317701641. Full Text.

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