Caspase 13 FLICA™ Kit
Quantitate caspase 13 in vitro with FAM-LEED-FMK
ICT's FLICA caspase 13 detection assay is a simple yet accurate method to measure caspase 13 activity in whole cells. Simply add the FLICA caspase 13 reagent to the media and incubate. Cells containing active caspase 13 will fluoresce green. The kit contains:
- ICT's FLICA caspase 13 detection probe, FAM-LEED-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:
Click here to buy
Regular size, ~100 tests:
Click here to buy
Caspase 13 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 13 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 13 enzyme. Please call for details or technical questions at 800-829-3194.
ICT's caspase 13 inhibitor probe is comprised of 3 segments:
- a green fluorescent label, FAM (carboxyfluorescein)
- an inhibitor peptide sequence targeted by active caspase 13 (LEED)
- 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.
Jurkat cells were treated with staurosporine, an apoptosis-inducing agent (bottom), or DMSO, a negative control (top), for 4 hours, then labeled with ICT’s green caspase 13 inhibitor probe, FAM-LEED-FMK, for 1 hour and read on a flow cytometer. Treatment with staurosporine induced caspase 13 activity in 92.7% of the experimental cells (M2, bottom right), whereas the negative control treatment exhibited caspase 13 activity in only 5.4% of the cell population (M2, top right). This is a ratio of 17:1. (Dr. Brian W. Lee, ICT).
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.