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 Caspase-1 probe allows researchers to assess caspase-1 activation.
The FLICA reagent 660-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because the 660-YVAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound 660-YVAD-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-1 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 Cellular 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. Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells.
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.
Jurkat cells were divided into treated and untreated pools and stained with 660-YVAD-FMK. After spiking FLICA into the cell culture media, Jurkat cells were treated with a negative control (left) or Nigericin (20 µM, catalog #6698) to induce caspase-1 activity (right) for 24 hours. Cells were washed 3 times and read on an Accuri C6 flow cytometer. Treatment with the negative control induced caspase-1 activity in only 6.8% of the cell population (left), whereas treatment with Nigericin induced caspase-1 activity in 57.3% of the experimental cells (right). This is a ratio of 8:1.
• Reagent name: 660-YVAD-FMK
• Target: Caspase-1
• 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 Caspase-1 Reagent (660-YVAD-FMK), 1 vial, #6323
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Kit Manual