FLICA® is a powerful method to assess cell death by detecting apoptosis in vitro. 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 turn-over of phosphatidyl serine, and eliminates the incidence of false positives that often plagues methods like Annexin V and TUNEL staining.
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 red fluorescent signal within the cell. Unbound FLICA® will diffuse out of the cell during the wash steps. Apoptotic cells will retain a higher concentration of FLICA® and fluoresce brighter than non-apoptotic cells. There is no interference from pro-caspases or inactive forms of the enzymes. If the treatment is causing cell death via apoptosis, apoptotic cells will have an elevated level of caspase activity relative to non- apoptotic or 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 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 #9145 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 red fluorescent label, sulforhodamine B (SR), and a fluoromethyl ketone (FMK). A caspase enzyme cannot cleave the FLICA® inhibitor probe; instead, it forms an irreversible covalent bond with the FMK on the target sequence and becomes inhibited from further enzymatic activity.
We offer four red SR-FLICA® inhibitors: SR-VAD-FMK, a poly-caspase probe, which may be used as a general reagent to detect apoptosis as it is recognized by all different types of activated caspases; SR- DEVD-FMK, which is preferred by active caspase-3/7; SR-LETD-FMK, which is preferred by active caspase-8; and SR-LEHD-FMK, which is preferred by active caspase-9. We also offer FLICA® inhibitor reagents with a green or far-red label (see our website for more details). Caspases, like most other crucial cell survival enzymes, are somewhat permissive in the target amino acid sequence they will recognize and cleave. 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 SR-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 green FAM dye will be quenched during the paraffin embedding process.
Cells labeled with SR-FLICA® can be counter-stained with reagents such as the live/dead stain 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 fluorescence plate reader or flow cytometer. For best results with flow cytometry, use a yellow or green laser. SR-FLICA® excites at 550-580 nm and emits at 590-600 nm.
- Prepare samples and controls.
- Dilute 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 – 1:60. For example, to stain at 1:30, add 10 µL to 290 µL of cultured cells. To stain at 1:60, add 5 µL to 295 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, 7-AAD, or an antibody.
- If desired, fix or embed cells.
- Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. SR-FLICA excites at 550-580 nm and emits at 590-600 nm.
Product Specific References
|Seredinski, S., et al. 2022. DNA topoisomerase inhibition with the HIF inhibitor acriflavine promotes the transcription of lncRNAs in endothelial cells. Molecular Therapy - Nucleic Acids, 1023-1035.