FLICA™ Caspase 8 Detection Kit
Quantitate caspase 8 in vitro with FAM-LETD-FMK
ICT's FLICA caspase 8 detection assay is a simple yet accurate method to measure caspase 8 activity in whole cells. Simply add the FLICA caspase 8 reagent to the media and incubate. Cells containing active caspase 8 will fluoresce green. The kit contains:
- ICT's FLICA caspase 8 detection probe, FAM-LETD-FMK
- 10x apoptosis wash buffer
- Hoechst stain (a blue DNA stain)
- Propidium iodide (a red live/dead stain)
- Fixative (for use after labeling with FLICA)
Trial size, ~25 tests:
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Regular size, ~100 tests:
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Caspase 8 activity may be detected in individual cells or suspensions of up to 1x10^6 cells/mL. The noncytotoxic FLICA probe is cell permeant: no lysis or permeabilization steps are necessary. Just culture cells, add FLICA to the media, incubate, and wash. Active caspase 8 enzymes will bind to FLICA and retain it within the cells, thereby capturing the green fluorescent signal. FLICA constantly fluoresces, therefore any unbound reagent must be washed out of the cell to remove any background noise. The resulting positive fluorescent signal can be detected with a fluorescence microscope, a fluorescence plate reader, or a flow cytometer. The green FLICA probe, FAM, excites at 490nm and emits at 520nm.
The FLICA assay also works with adherent cells and very thin tissue sections. FLICA will not work in previously fixed cells or paraffin cells, as those treatments inactivate the caspase 8 enzyme. Please call for details or technical questions at 800-829-3194.
ICT's caspase 8 inhibitor probe is comprised of 3 segments:
- a green fluorescent label, FAM (carboxyfluorescein)
- an inhibitor peptide sequence targeted by active caspase 8 (LETD)
- and a fluoromethylketone group (FMK), which forms a covalent bond with the active enzyme.
FLICA kits have been used successfully with a multitude of cell lines from a variety of species (human, mouse, rat, etc), including, but not limited to, cancer cells, neurons, primary cells, epithelial cells, endothelial cells, and macrophages. For in vitro apoptosis detection via active poly caspases, see our FLICA Poly Caspases Detection Kits.
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 ug/mL cycloheximide (CHX) for 24 hours. Cells were then labeled 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).
Sample protocol:
- Culture your cells up to 1 x 10^6 cells/mL.
- Induce apoptosis following your protocol, and create positive and negative controls.
- Reconstitute the reagent with 50mcL DMSO to form the stock concentrate (which can be frozen for future use).
- Dilute the stock concentrate with 200mcL 1X PBS to form the working solution.
- Add ~10mcL of the working solution directly to a 300-500mcL aliquot of your cell culture for labeling.
- Incubate 1-4 hours.
- Wash and spin cells twice, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer.
- If desired, label cells with Hoechst stain.
- If desired, label cells with Propidium Iodide or 7AAD.
- If desired, fix cells.
- Analyze data using a fluorescence microscope, plate reader, or flow cytometer.