Pyroptosis 660 Caspase-1
Jurkats were stained with Pyroptosis 660 Caspase-1 Kit for two hours followed by treatment with 10 μM Nigericin as shown. Pyroptosis could be detected by an increase in active Caspase-1 detected by an increase in 660 far red fluorescence. Data analyzed on the ZE5 Cell Analyzer
Pyroptosis 660 Caspase-1 Kit utilizes the popular FLICA technology to detect caspase-1 activation. This kits contain the caspase-1 inhibitor reagent YVAD-FMK, which has the preferred binding sequence for caspase-1, Tyr-Val-Ala-Asp (YVAD) (Chapman, 1992). This preferred caspase-1 binding sequence is labeled with 660 a far red fluorescent dye and linked to a fluoromethyl ketone (FMK) reactive entity. Caspase-1 will not cleave the FLICA inhibitor probe; instead, it forms an irreversible covalent bond with the FMK group on the reagent and becomes inhibited from further enzymatic activity.
Product Details
- Reagents in the Kit
- 1 vial of 660-YVAD-FMK caspase-1 inhibitor - lyophilized
1 vial Nigericin
10x Cellular Wash Buffer, 15 mL
Fixative, 6 mL
1 vial Hoechst Stain, 1 ml - Test Principle
- To use FLICA, add directly to the cell culture medium, incubate, and wash. FLICA is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase-1 enzyme inside the cell, it will covalently bind with YVAD-FMK and retain the fluorescent signal within the cell. Unbound FLICA will diffuse out of the cell during the subsequent wash steps. Therefore, positive cells will retain a higher concentration of FLICA and fluoresce brighter than negative cells. There is no interference from pro-caspases or inactive forms of the enzymes. After labeling with FLICA, cells can be counter-stained with other reagents and fixed or frozen. Cells labeled with YVAD-FMK can be counter-stained with reagents such as the red live/dead stains Propidium Iodide and 7-AAD. Nuclear morphology may be concurrently observed using Hoechst 33342 (included in the kit), a blue DNA-binding dye. Cells can be viewed through a fluorescence microscope or flow cytometer.
Storage Information
- Storage
- MULTIPLE STORAGE CONDITIONS APPLY ON ARRIVAL. Store the unopened kit (and each unopened component) according to the storage instructions on each component label. Store the Nigericin at -20oC. Once reconstituted, the Nigericin stock should be used immediately or alliquoted and stored at -20oC for 12 months. Avoid repeated freezing and thawing.
- Guarantee
- Guaranteed until date of expiry. Please see product label.
More Information
- Acknowledgements
- FLICA is a trademark of Immunochemistry Technologies, LLC.
- Regulatory
- For research purposes only
Applications of Pyroptosis 660 Caspase-1
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| Flow Cytometry | Refer to Instructions For Use | ||
| Immunofluorescence |
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
- Instructions For Use
- Instructions for use can be found at https://www.bio-rad-antibodies.com/static/uploads/ifu/ict9158.pdf
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