Pyroptosis/Caspase-1 Assay, Green

$304.00$597.00

ICT’s Pyroptosis/Caspase-1 Assay, Green utilizes our popular FLICA technology to measure caspase-1 activation. Using this kit, researchers can easily detect the pyroptotic cells in their whole cell samples in vitro. This complete kit includes Nigericin for the simple and reliable generation of a positive control. Analyze the green fluorescent signal using fluorescence microscopy, a fluorescence plate reader, or by flow cytometry.

Catalog # Size Quantity Price
9145 25 - 50 Tests $304.00
9146 100 - 200 Tests $597.00
Catalog #: 9145, 9146 Categories: , ,

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, Green can be used by researchers to assess caspase-1 activity in pyroptotic cells.

This kit utilizes the green, cell permeant FLICA reagent, FAM-YVAD-FMK, for the in vitro detection of caspase-1 in whole living cells. FAM-YVAD-FMK enters each cell and irreversibly binds to activated caspase-1. Because FAM-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 green 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 plate reader, 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 Propidium Iodide, 7-AAD, or Hoechst 33342 (included in the kit). 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 FAM-FLICA with 50 µL DMSO.
4. Dilute FAM-FLICA 1:5 by adding 200 µL PBS.
5. Add diluted FAM-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, Propidium Iodide, 7-AAD, or an antibody.
10. If desired, fix cells.
11. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excited at 492 nm and emits at 520 nm.

THP-1 suspension cells were treated with PMA (5 ng/mL) to induce differentiation into macrophages. After 48 hours, PMA was removed and replaced with fresh medium that was spiked with LPS (10 ng/mL) to induce caspase-1 activation. After 2 hours, cells were stained with FAM-YVAD-FMK for 1 hour, washed, and examined under a fluorescence microscope. The top images reveal many experimental cells, several of which fluoresce green, therefore they have active caspase-1. The lower right image reveals many control cells in the field of view, however the corresponding fluorescence image is dark (lower left); few of these cells have active caspase-1. Data courtesy of Dr. Brian Lee, ICT.

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• Reagent name: FAM-YVAD-FMK
• Excitation/Emission: 492 nm / 520 nm
• Method of Analysis: Flow cytometer, fluorescence microscope, fluorescence plate reader
• Types of Samples: Cell culture
• Storage: ≤ -20°C for Nigericin component, 2-8°C for remaining kit components
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 9145: 25-50 Tests
• FLICA Caspase-1 Inhibitor Reagent (FAM-YVAD-FMK), 1 vial, #655
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Nigericin, 0.5 µmoles, #6698
• Kit Manual

Kit 9146: 100-200 Tests
• FLICA Caspase-1 Inhibitor Reagent (FAM-YVAD-FMK), 4 vials, #655
• 10X Cellular Wash Buffer, 60 mL, #6165
• Fixative, 6 mL, #636
• Hoechst 33342, 1 mL, #639
• Nigericin, 0.5 µmoles, #6698
• Kit Manual

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