Inflammatory caspases, such as caspase-1, play a critical role in the mechanism to trigger the lytic programmed cell death known as pyroptosis. ICT’s Pyroptosis/Caspase-1 Assay, Far Red can be used by researchers to assess caspase-1 activity in pyroptotic cells.
This kit utilizes the far red, cell permeant FLICA reagent, 660-YVAD-FMK, for the in vitro detection of caspase-1 in whole living cells. 660-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because 660-YVAD-FMK becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound 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 a fluorescence microscope or flow cytometer. Cells labeled with the FLICA reagent may be read immediately, or preserved for up to 16 hours using Fixative (included in the kit). Unfixed samples may also be counter-stained with Hoechst 33342 (included in the kit) to label cell nuclei. This complete kit also includes Nigericin as a convenient option for generating a positive control. Nigericin induces a net decrease in intracellular levels of potassium, crucial for activation of caspase-1. In pyroptosis experiments, it has been shown to generate robust caspase-1 activation in a variety of cell types.
1. Prepare samples and controls.
2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O.
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 sample).
6. Incubate approximately 1 hour.
7. Remove media and wash cells 3 times: add 1X Cellular Wash Buffer and spin cells.
8. Resuspend cell pellet in 1X Cellular Wash Buffer.
9. If desired, label with additional stains, such as Hoechst 33342, DAPI, or an antibody.
10. If desired, fix cells.
11. Analyze with a fluorescence microscope or flow cytometer. FLICA 660 is excited at 660 nm and emits at 680-690 nm.
Caspase-1 activity in nigericin-treated Jurkat cells. Jurkat cells were mock treated with a negative control (Non-Induced, Panels A-C), or Nigericin (10 µM) to induce NLRP3 inflammasome and caspase-1 activation (Nigericin, Panels D-F). Samples were then immediately stained with 660-YVAD-FMK, therefore FLICA reagent was present throughout the induction process. Following addition of FLICA, the cells were incubated for 24 hours at 37°C, washed, stained with Hoechst 33342 for 15 minutes at room temperature (to label nuclei blue), and examined under a Nikon Eclipse 90i fluorescence microscope equipped with a Hamamatsu Flash 4.0 camera. In the non-induced sample, many cells with blue nuclei are visible in Panel B. However, in Panel A, which shows 660-YVAD-FMK labeling, no red cells with active casapse-1 are visible. Panel C shows the overlay image combining the blue and red fluorescence channels with the corresponding differential interference contract (DIC) image, which reveals cell morphology. In the Nigericin treated sample, many cells with blue nuclei are visible in Panel E, the majority of which are labeled red in Panel D, indicating the presence of active caspase-1 enzymes. Panel F shows the image made by overlaying blue fluorescence, red fluorescence, and DIC channels into a single combined image.
• 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
9158 – 25 Tests:
• 1 vial of 660-YVAD-FMK caspase-1 inhibitor reagent, #6323
• 1 bottle of 10X Cellular Wash Buffer (15 mL), #6164
• 1 bottle of Fixative (6 mL), #636
• 1 vial of Hoechst 33342, 200 μg/mL (1 mL), #639
• 1 vial of Nigericin, 0.5 μmoles, #6698