FLICA Apoptosis Detection Kits

FLICA™ in vitro Apoptosis Detection Kits
Detect caspase activity in whole living cells.
Distinguish apoptosis from necrosis.

ICT's FLICA™ kits make it easy to measure active caspases in whole living cells: apoptotic cells fluoresce red or green. Use FLICA™ to quantitate apoptosis, to distinguish apoptosis from necrosis, and measure cytotoxicity. Detect all caspases at once using the poly caspases FLICA™ kits, or detect specific caspase 1, 2, 3&7, 6, 8, 9, 10, or 13 using ICT’s specialized FLICA™ kits (prices).

Normal and keratoconus fibroblasts labeled with Caspase 3 FLICA, catalog # 93

FLICA™ kits are not ELISAs and they do not use antibodies for detection. Instead, they use an inhibitor sequence of caspases (such as VAD, which reacts with all caspases) linked to a green (carboxyfluorescein, FAM) or red (sulforhodamine, SR) fluorescent probe. FLICA™= Fluorescent-Labeled Inhibitor of CAspases.
FLICA™ kits are fairly simple to use and experiments can be completed in 1 day. The reagents are cell-permeant, so you don't have to lyse the cells or

Normal (left) and keratoconus (right) corneal fibroblasts were treated with 200uM H2O2 for 1hr, washed, and allowed to recover for 1-3hrs. The culture media was removed and replaced with 1x FAM-FLICA™ Caspases 3&7 (kit cat.# 93) solution (in culture media) at 300ul/well for 1hr. 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.

permeabilize the membranes. Just add FLICA™ to your cell culture media, let it incubate 1-4 hours, wash the cells, and analyze with a fluorescence microscope, plate reader, or flow cytometer.
     Once added to the media of your cell culture, FLICA™ will pass through the cell wall. If there is an active caspase enzyme inside the cell, it will form a covalent bond with FLICA™ and retain the green or red fluorescent signal within the cell. Because the caspase enzyme itself binds with FLICA™, only active caspases will be measured. There is no interference from pro-caspases nor inactive forms of the enzyme. FLICA™ probes constantly fluoresce, therefore, any unbound reagent must be washed back out of the cell to remove any background fluorescence. This is done by several quick rinse and spin steps, or further incubation with the wash buffer or media.
     Add a necrotic stain, like PI (which is included in the green FAM kits) or 7-AAD to distinguish apoptotic cells from necrotic cells. To label DNA, Hoechst 33342 is included in all FLICA™ kits. Once labeled with FLICA™, cells can be fixed, embedded, or frozen for storage (a fixative is included).
     Read the fluorescent signal with a microscope, plate reader, or flow cytometer. Green FAM-FLICA™ excites at ~490nm and emits at 530nm (FAM manual). Red SR FLICA™ excites at ~560 and emits at 590 (SR manual).
     FLICA™ works with suspension cells, adherent cells, thin tissue sections, and thin frozen sections from human, monkey, chicken, mouse, rat, drosophila, yeast, and paramecia. FLICA™ kits have been used successfully with THP-1, HL60, MCF7, HeLa, Jurkat, epithelial cells, retinal cells, primary neurons, macrophages, lymphocytes, and fibroblasts, to name a few (list of cell types). To image apoptotic cells in live animals in vivo, use ICT's FLIVO™ kits.
    FLICA™ will NOT work with fixed nor paraffin embedded cells, as the caspase enzymes have become inactivated. However, cells can be fixed, embedded, or frozen after labeling with FLICA™ (a fixative is included). We recommend reading within 24 hours, as the fluorescent label may photobleach, however samples have been frozen for 8 weeks and re-analyzed with equivalent results.
     FLICA™ kits come in 2 sizes: 25 tests and 100 tests. For example, 1 test is a 300uL aliquot of cells grown at 1X10⁶ cells/mL and analyzed on a fluorescence plate reader. Plate readers tend to require the most reagent, flow cytometers the least. Set up an initial titration experiment to determine of the amount of reagent and washing procedure that will work best for your experimental conditions. Create positive controls by inducing apoptosis with ICT's camptothecin or staurosporine.
     FLICA™ makes it simple to identify apoptotic cells. To detect apoptosis in general, use the green or red poly caspases kits. These kits use a general peptide sequence (VAD) which reacts with all active caspases. To distinguish apoptotic from necrotic cells, use ICT's green FAM-FLICA™ kit to label apoptotic cells green, then add PI (included with the kit) or 7-AAD to stain necrotic cells red.
     The best way to assess the total health of cultured cells via flow cytometry is to combine ICT's green fluorescent poly-caspases FAM-VAD-FMK FLICA reagent with 7AAD, a red fluorescent necrotic stain. To use, just take a small aliquot of cells, add a low dose of FAM-FLICA, let that incubate 30”, add 7AAD for 5”, then read on a flow cytometer to quantitate living, dead, and apoptotic cells. This procedure requires no wash steps, as the sheathing fluid running through the flow cytometer acts as a wash step to remove any unbound probe.
     To target a specific caspase, use ICT’s specialized FLICA™ kits for caspase 1, 2, 3&7, 6, 8, 9, 10, or 13. All kits are available with a green fluorescent probe. 3 kits are available with a red fluorescent probe: poly caspases, caspases 3&7, and caspase 9. Use the red SR-FLICA™ kits with GFP transfected cell lines and for dual-labeling studies with other green reagents. Add a colored secondary antibody to generate more data with 1 experiment.

Don't set up another apoptosis experiment until you call us. Order today 800-829-3194!

Pan Caspases FLICA Apoptosis Detection kit, catalog # 91 Green FLICA™ kits contain: FAM-FLICA™ reagent, apoptosis wash buffer, fixative, Hoechst 33342, and propidium iodide. Cat.# 91, 25 tests, left.

Pan Caspases FLICA Apoptosis Detection kit, catalog # 917 Red FLICA™ kits contain: SR-FLICA™reagent, apoptosis wash buffer, fixative, and Hoechst 33342. Cat.# 917, 100 tests, left.

More reliable than annexin.
FLICA™ kits are more accurate and reliable than annexin. Annexin detects phosphatidyl serine which is exposed upon the turnover of the cell membrane. This is not an accurate indicator of apoptosis as this turnover can occur for other reasons. Annexin tends to bind to all thymus-derived cells, apoptotic or not.

Earlier and easier than TUNEL.
ICT's kits are easier and more sensitive than TUNEL. TUNEL is a long procedure based on DNA laddering. FLICA™ kits detect apoptosis based on caspase activity, which occurs before DNA laddering, so you get an earlier indicator of apoptosis.

FLICA™ Kits:
• Easy Just add the reagent directly to the cell culture and incubate.
• Fast The reactions start within 15 minutes of addition to the cells,
however we recommend an incubation of 1-4 hours.
• Accurate There is no interference from pro-caspases or inactive forms of the enzyme.
• Reliable Only cells with active caspase enzymes fluoresce.
• Sensitive Detects background levels of apoptosis in controls.
• Quantitative Read with a fluorescence plate reader, microscope, or flow cytometer.
• Use whole living cells Study apoptosis as it occurs in the cell.
• No lysis or permeabilization FLICA™ is cell-permeant; you don’t have to destroy the cell to study it.
• Do not kill the cells The reagents may be incubated up to 72 hours without killing the cells.
• Do not use antibodies FLICA™ uses an inhibitor peptide sequence linked to a green or red fluorescent label.
• Detect apoptosis earlier than Annexin V and TUNEL
Caspases are released before the phospholipid turnover & DNA laddering.

Data:
• Cell lines successfully labeled with FLICA™.
• Apoptosis - learn about it in our slide show with FLICA™-labeling and PI.
• FLICA™ detects neurotoxicity in primary neurons (microscope data).
• Caspases revealed in yeast using SR-FLICA™ (FACS).
• Detect caspases and serine proteases concurrently using flow cytometry.
• Stathmoapoptosis in HL60 cells.
• Data and protocol in our green FAM-FLICA manual.
• Data and protocol in our red SR-FLICA manual.
• Data in our newsletter.
• Tumor cells examples.
• Handout on tumor applications.

Sample protocol:
1. Culture your cells up to 1 x 10⁶ cells/mL.
2. Induce apoptosis following your protocol, and create positive and negative controls.
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which can be frozen for future use).
4. Dilute the stock concentrate with 200uL 1X PBS to form the working solution.
5. Add ~10uL of the working solution directly to a 300-500uL 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.