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Pyroptosis 660 Caspase-1

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Detection of pyroptosis in Jurkats

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

Product Type
Kits
Specificity
Pyroptosis 660 Caspase-1

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ICT9158
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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.

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.
Max Ex/Em
Fluorophore Excitation Max (nm) Emission Max (nm)
FLICA 660 660 685
Regulatory
For research purposes only
Guarantee
Guaranteed until date of expiry. Please see product label.
Acknowledgements
FLICA is a trademark of Immunochemistry Technologies, LLC.

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.

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
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
ICT9158
Instructions for use can be found at https://www.bio-rad-antibodies.com/static/uploads/ifu/ict9158.pdf
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