FLICA™ Caspases 3 & 7 Kits

Quantitate caspases 3&7 in vitro with FAM-DEVD-FMK and SR-DEVD-FMK

ICT's FLICA caspases 3&7 detection assays are simple yet accurate methods to measure the activity of caspases 3&7 in whole cells. Simply add the FLICA caspases 3&7 reagent to the media and incubate. Cells containing active caspases 3&7 will fluoresce green or red.

Green FLICA Caspases 3&7 Detection kit contains:

  • ICT's green FLICA caspases 3&7 detection probe, FAM-DEVD-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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Red FLICA Caspases 3&7 Detection kit contains:

Trial size, ~25 tests:
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Regular size, ~100 tests:
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Use ICT's green or red FLICA™ Caspase 3 & 7 Detection Kits for apoptosis detection via caspase 3 & 7 activity in whole, living cells. FLICA™ Caspase 3 & 7 Detection Kits (Fluorescent-Labeled Inhibitors of CAspases) are based on an inhibitor peptide sequence (DEVD) targeted by both caspase 3 and caspase 7. This sequence is linked to a green or red fluorescent probe. The green probe is carboxyfluorescein (FAM), which is brighter and more cell-permeant than FITC. The red probe is sulforhodamine B (SR). The probes also contain a fluoromethyl ketone group (FMK), which forms a covalent bond with the active caspase enzyme.

Because of the non-polar nature and relatively small molecular weight of these FLICA probes, they are cell-permeant. FLICA easily crosses the cell membrane, negating any need for lysis or membrane permeabilization steps. The detection assay is easy - just add FLICA to the media and incubate between 1-4 hours. Active caspase 3 & 7 enzymes will form covalent bonds with the FLICA inhibitor probe. Once bound to an enzyme, the fluorescent reagent is retained within the cell. The binding event prevents the caspase from further catalysis but will not stop apoptosis from proceeding. The reagent will start to react with active caspase 3 & 7 enzymes within 15 minutes of addition to the media.

FLICA reagents continually fluorescence, whether bound to a caspase or not. Therefore, a simple wash and spin step must be done after the reagent incubation period to remove any unbound reagent (there may be some background noise from unbound reagent, but this is very low). FLICA is so sensitive that it will even detect innate apoptosis in non-induced cells, which is typically 2-6% of all cells. FLICA is specific for active caspases - it will not react with pro-caspases nor inactive forms of the enzyme. Cells can be fixed or paraffin-embedded after labeling with FLICA. Green FAM- FLICA excites at around 490nm and emits at 530nm; red SR- FLICA excites at 560 and emits at 590. Read cells with a fluorescence microscope, plate reader, or flow cytometer.

The activity of caspases 3&7 may be detected in individual cells or suspensions of up to 1x10^6 cells/mL. The noncytotoxic FLICA probes are cell permeant: no lysis or permeabilization steps are necessary. Just culture cells, add FLICA to the media, incubate, and wash. Active caspases 3&7 enzymes will bind to FLICA and retain it within the cells, thereby capturing the green or red 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 red FLICA probe, SR, excites at 560nm and emits at 590nm.

The FLICA assays will also work with adherent cells and very thin tissue sections. FLICA will not work in previously fixed cells or paraffin cells, as those treatments inactivate the caspases 3&7 enzymes. Please call for details or technical questions at 800-829-3194.

ICT's caspases 3&7 inhibitor probes are comprised of 3 segments:

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.

Mouse EL4 thymoma cells were cultured in the absence (A) or presence (B) of 100ug mitomycin for 18 hours. The cells were harvested and stained with ICT’s SR-DEVD-FMK inhibitor probe (red histogram) to detect active caspases 3&7. The data illustrate a low level of caspase activity in untreated cells (panel A) compared to unstained control (blue histogram) that is greatly enhanced in the mitomycin treated cultures (panel B). Cells were analyzed with a BD LSRII flow cytometer to detect sulforhodamine using the green laser (532nm) and detector D set up with filter 610/620 600LP (Dr. Catherine Martone, Yale).

caspases 3&7 flow cytometry data

Caspases 3&7 positive cells

Normal (left) and keratoconus (right) corneal fibroblasts were treated with 200uM H2O2 for 1 hour, washed, and allowed to recover for 1-3 hours. The culture media was removed and replaced with 1x FAM-FLICA™ Caspases 3&7 (FAM-DEVD-FMK) solution (in culture media) at 300ul/well for 1 hour. The cell layer was washed 3 times with 1x wash buffer; 300ul wash buffer was added to keep the cells from drying. Keratoconus corneal fibroblasts treated with H2O2 (right) show a significant increase in caspases 3&7 activity compared to normal cells (left). Non-apoptotic cells are dark in background. Data courtesy of Dr. Cristina Kenney, M.D., Ph.D. Dept. of Ophthalmology, UC Irvine.

Sample protocol:

  1. Culture your cells up to 1 x 10^6 cells/mL.
  2. Induce apoptosis following your protocol, and create positive and negative controls.
  3. Reconstitute the reagent with 50mcL DMSO to form the stock concentrate (which can be frozen for future use).
  4. Dilute the stock concentrate with 200mcL 1X PBS to form the working solution.
  5. Add ~10mcL of the working solution directly to a 300-500mcL aliquot of your cell culture for labeling.
  6. Incubate 1-4 hours.
  7. Wash and spin cells twice, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer.
  8. If desired, label cells with Hoechst stain.
  9. If desired, label cells with Propidium Iodide or 7AAD.
  10. If desired, fix cells.
  11. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.