Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells. The FLICA 660 Poly Caspase probe allows researchers to assess caspase activation.
The FLICA reagent 660-VAD-FMK enters each cell and irreversibly binds to activated caspases. Because the 660-VAD-FMK FLICA reagent becomes covalently coupled to the active enzymes, it is retained within the cell, while any unbound 660-VAD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining far red fluorescent signal is a direct measure of the active caspase enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by fluorescence microscopy or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative. Unfixed samples may also be analyzed with Hoechst stain to detect changes in nuclear morphology.
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.
To demonstrate the fluorescence properties and apoptosis detection capability of the new far-red fluorescent FLICA 660 Poly Caspase Assay, Jurkat cells were were treated with 1 µM staurosporine for 4 hours and labeled with FLICA 660-VAD-FMK poly caspase inhibitor reagent.
Using a Nikon E800 Hyperspectral Microscope equipped with a Photometrics HQ2 camera, far-red fluorescence emission from apoptotic Jurkat cells was acquired with a Cy5 HYQ bandpass filter combination set. FLICA 660-VAD-FMK reagent optimally excites at 660 nm and has an emission max at 690 nm, making it suitable for use with red lasers on common benchtop flow cytometers and ideal for dual staining purposes with green fluorescent probes.
Composite overlay of DIC image and far-red fluorescence emission from apoptosis-induced Jurkat cells stained with FLICA 660 Poly Caspase Assay.
• Reagent name: 660-VAD-FMK
• Target: Poly caspases
• Excitation/Emission: 660 nm / 690 nm
• Method of Analysis: Flow Cytometer, Fluorescence Microscope
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• FLICA Poly Caspase Reagent (660-VAD-FMK), 1 vial, #6322
• 10X Apoptosis Wash Buffer, 15 mL, #635
• Fixative, 6 mL, #636
• Kit Manual