Far-Red Fluorescent FLICA® 660 Poly (active) Caspase (VAD) Assay Kit

This in vitro assay employs the far-red fluorescent inhibitor probe 660-VAD-FMK to label active caspase enzymes in living cells. Analyze the fluorescent signal using fluorescence microscopy or flow cytometry.



SKU: 9120

Size: 25-50 Tests
Price:
Sale price$257

FLICA® is a powerful method to assess caspase activity. FLICA® probes are cell permeant, noncytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind with active caspase enzymes. We have developed a far-red excitation and emission spectra FLICA® 660 probe for the detection of cells bearing active caspases.

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. 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. 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.

Our FLICA® 660 poly caspase inhibitor probe contains the preferred binding sequence for all caspases, Val-Ala-Asp (VAD). This preferred poly caspase tripeptide binding sequence is labeled at the amino terminus end with a far-red fluorescent 660 dye and linked at the carboxyl end to a fluoromethyl ketone (FMK) reactive entity. The resulting cell permeant, fluorescent molecule, 660-VAD-FMK, optimally excites at 660 nm and emits between 685-690 nm. A conventional red HeNe laser with a 633 nm excitation provides excellent excitation efficiency, enabling cells labeled with FLICA 660 to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities.

To use FLICA® , add it directly to the cell 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® 660-VAD-FMK and retain the far-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 enzyme. 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® . After labeling with FLICA® , cells can be counter-stained with other reagents and fixed or frozen.

660-VAD-FMK
Poly caspases
660 nm / 690 nm
Flow cytometry, Fluorescence microscope
Cell culture
2-8°C
Domestic: Overnight Delivery; International: Priority Shipping
United States
  1. Prepare samples and controls
  2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
  3. Reconstitute FLICA with 50 µL DMSO.
  4. Dilute FLICA 1:5 by adding 200 µL PBS.
  5. Add diluted FLICA to each sample at 1:30-1:60 (e.g. spike at 1:30 by adding 10 µL to 290 µL cultured cells).
  6. Incubate approximately 1 hour.
  7. Wash and spin cells three times.
  8. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.
  9. If desired, fix cells.
  10. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 excites at 660 nm and emits at 680-690 nm.
  • FLICA Poly Caspase Reagent (660-VAD-FMK), 1 vial, #6322
  • 10X Apoptosis Wash Buffer, 15 mL, #635
  • Fixative, 6 mL, #636
  • Kit Manual
  • Elsherbini A, Kirov AS, Dinkins MB, Wang G, Qin H, Zhu Z, Tripathi P, Crivelli SM, Bieberich E. Association of Aβ with ceramide-enriched astrosomes mediates Aβ neurotoxicity. Acta Neuropathol Commun. 2020 Apr 28;8(1):60. doi: 10.1186/s40478-020-00931-8. Full Text

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