Our FLICA® probes are non-cytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind to active caspase enzymes. FLICA® measures the intracellular process of apoptosis instead of a side-effect, such as the turnover of phosphatidyl serine, and eliminates the incidence of false positives that often plagues methods like Annexin V and TUNEL staining. FLICA® can also be used to measure pyroptosis, a highly inflammatory form of programmed cell death. To use FLICA®, add it directly to the cell culture media, incubate, and wash. FLICA® is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase enzyme inside the cell, it will covalently bind with FLICA® and retain the green fluorescent signal within the cell. Unbound FLICA® will diffuse out of the cell during the wash steps. Apoptotic and pyroptotic cells will retain a higher concentration of FLICA® and fluoresce brighter than healthy cells. There is no interference from pro-caspases or inactive forms of the enzymes. If the test treatment is causing cell death via apoptosis and/or pyroptosis, the cells will contain an elevated level of caspase activity relative to negative control cells and fluoresce with FLICA®. Apoptosis is an evolutionarily conserved process of programmed cell suicide. It is centered on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Once activated, caspases cleave protein substrates leading to the eventual disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Mammalian caspases play distinct roles in both apoptosis and inflammation. In apoptosis, effector caspases (-3, -6, and -7) are responsible for proteolytic cleavages that lead to cell disassembly. Initiator caspases (-8, -9, and -10) regulate apoptosis upstream. Caspase-1 is associated with pyroptosis and inflammasome activity and takes on the role of a key housekeeping enzyme in its conversion of pro-IL-1ß protein into the active IL-1ß cytokine (Use FLICA® kits #98, #9122, and #9162 to detect caspase-1). Please note that macrophages and monocytes have been shown to rapidly secrete caspase-1 upon activation. Like the majority of other proteases, caspases are synthesized as pro-form precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity. Active caspase enzymes consist of two large (~20 kD) and two small (~10 kD) subunits that non-covalently associate to form a two heterodimer, tetrameric active caspase.
Activated caspase enzymes cleave proteins by recognizing a 3 or 4 amino acid sequence that must include an aspartic acid (D) residue in the P1 position. This C–terminal residue is the target for the cleavage reaction at the carbonyl end. Each FLICA® probe contains a 3 or 4 amino acid sequence that is targeted by different activated caspases. This target sequence is sandwiched between a green fluorescent label, carboxyfluorescein (FAM), and a fluoromethyl ketone (FMK). Caspases cannot cleave the FLICA® inhibitor probe; instead, they form an irreversible covalent bond with the FMK on the target sequence and enzyme activity is inhibited. Our poly caspase FLICA® probe, FAM-VAD-FMK, can be used as a general reagent to detect apoptosis as it is recognized by many types of activated caspases. To more specifically target a particular caspase enzyme, use one of our specialized FLICA® reagents. We have kits for the detection of: caspase-1 (YVAD or WEHD) (also recognizes caspases 4 and 5), -2 (VDVAD), -3/7 (DEVD), -6 (VEID), -8 (LETD), -9 (LEHD), and -10 (AEVD). FLICA® kits are also available with a red or far red fluorescent label. Caspases, like most other crucial cell survival enzymes, are somewhat permissive in the target amino acid sequence they will recognize and cleave. Therefore, although FLICA® reagents contain the different amino acid target sequences preferred by each caspase, they can also recognize other active caspases when they are present. We encourage validation of caspase activity by an orthogonal technique. FLICA® can be used to label suspension or adherent cells and thin tissue sections. After labeling with FAM-FLICA®, cells can be fixed or frozen. For tissues that will be paraffin-embedded after labeling, use our red sulforhodamine SR-FLICA® probes; do not use the green FAM-FLICA® probes as the FAM dye will be quenched during the paraffin embedding process. Cells labeled with FAM-FLICA® can be counter-stained with reagents such as the red live/dead stains Propidium Iodide (included in FAM-FLICA® kits) and 7-AAD (catalog # 6163) to distinguish apoptosis from necrosis. Nuclear morphology can be concurrently observed using Hoechst 33342, a blue DNA binding dye (included in FLICA® kits). Cells can be viewed directly through a fluorescence microscope, or the fluorescence intensity can be quantified using a flow cytometer or fluorescence plate reader. FAM-FLICA® optimally excites at 488-492 nm and has a peak emission at 515-535 nm.
- Prepare samples and controls
- Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
- Reconstitute FLICA with 50 µL DMSO.
- Dilute FLICA 1:5 by adding 200 µL PBS.
- Add diluted FLICA to each sample at 1:30 (e.g., add 10 µL to 290 µL of cultured cells).
- Incubate approximately 1 hour.
- Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.
- If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.
- If desired, fix cells.
- 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.
Product Specific References
PMID | Publication |
38788367 | Radomska, D., et al. 2024. Evaluation of anticancer activity of novel platinum(II) bis(thiosemicarbazone) complex against breast cancer. Bioorganic chemistry, 107486. |
37834182 | Scopelliti, F., et al. 2023. Functional TRPA1 Channels Regulate CD56dimCD16+ NK Cell Cytotoxicity against Tumor Cells. International journal of molecular sciences, . |
37001390 | Ivasechko, I., et al. 2023. Molecular design, synthesis and anticancer activity of new thiopyrano[2,3-d]thiazoles based on 5-hydroxy-1,4-naphthoquinone (juglone). European journal of medicinal chemistry, 115304. |
37128606 | Capitini, C., et al. 2023. APP and Bace1: Differential effect of cholesterol enrichment on processing and plasma membrane mobility. iScience, 106611. |
37200190 | Fike, A.J., et al. 2023. STAT3 signaling in B cells controls germinal center zone organization and recycling. Cell reports, 112512. |
37247053 | Wankell, M., et al. 2023. Testing Cell Migration, Invasion, Proliferation, and Apoptosis in Hepatic Stellate Cells. Methods in molecular biology (Clifton, N.J.), 43-54. |
37371034 | Rok, J., et al. 2023. The Assessment of Anti-Melanoma Potential of Tigecycline-Cellular and Molecular Studies of Cell Proliferation, Apoptosis and Autophagy on Amelanotic and Melanotic Melanoma Cells. Cells, . |
37553330 | Matsui, Y., et al. 2023. SNIP1 and PRC2 coordinate cell fates of neural progenitors during brain development. Nature communications, 4754. |
37748566 | Zhou, Ling, L., et al. 2023. Extracellular ATP (eATP) inhibits the progression of endometriosis and enhances the immune function of macrophages. Biochimica et biophysica acta. Molecular basis of disease, 166895. |
37477437 | Dong, Xijie, X., et al. 2023. INTRINSIC/EXTRINSIC APOPTOSIS AND PYROPTOSIS CONTRIBUTE TO THE SELECTIVE DEPLETION OF B CELL SUBSETS IN SEPTIC SHOCK PATIENTS. Shock (Augusta, Ga.), 345-353. |
37760225 | Pezo, Felipe, F., et al. 2023. Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests. Animals: an open access journal from MDPI, . |
35055021 | Rok, J., et al. 2022. The Anticancer Potential of Doxycycline and Minocycline-A Comparative Study on Amelanotic Melanoma Cell Lines. International journal of molecular sciences, . |
34994998 | Chiu, Y.J., et al. 2022. Curcumin suppresses cell proliferation and triggers apoptosis in vemurafenib-resistant melanoma cells by downregulating the EGFR signaling pathway. Environmental toxicology, . |
35111247 | Huang, C.F., et al. 2022. Quercetin induces tongue squamous cell carcinoma cell apoptosis via the JNK activation-regulated ERK/GSK-3α/β-mediated mitochondria-dependent apoptotic signaling pathway. Oncology letters, 78. |
35270004 | González-Sarrías, A., et al. 2022. Milk-Derived Exosomes as Nanocarriers to Deliver Curcumin and Resveratrol in Breast Tissue and Enhance Their Anticancer Activity. International journal of molecular sciences, . |
35241512 | Tsai, Y.F., et al. 2022. Gadodiamide Induced Autophagy and Apoptosis in Human Keratinocytes. In vivo (Athens, Greece), 603-609. |
35842415 | Trugilho, M.R.O., et al. 2022. Platelet proteome reveals features of cell death, antiviral response and viral replication in covid-20. Cell death discovery, 324. |
35939511 | Kawashima, A., et al. 2022. Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells. PLoS neglected tropical diseases, e0010672. |
35967457 | Su, M., et al. 2022. Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis. Nature cardiovascular research, 732-747. |
36063959 | Sun, Y., et al. 2022. A single-beam of light priming the immune responses and boosting cancer photoimmunotherapy. Journal of controlled release : official journal of the Controlled Release Society, 734-747. |
36296570 | Janowska, S., et al. 2022. Synthesis and Anticancer Activity of 1,3,4-Thiadiazoles with 3-Methoxyphenyl Substituent. Molecules, . |