Frequently Asked Questions
Caspase-Glo® 1 Inflammasome Assay
The complete protocol for this product is available in Technical Manual #TM456.
What type of multiwell plates should I use for this assay?
For optimum performance, use opaque, white multiwell plates that are compatible with your luminometer. Luminescence signal is diminished in black plates and increased well-to-well cross talk is observed in clear plates. RLU values, such as those shown in the figures of the Technical Bulletin, will vary depending on which plates and luminometers are used to generate data. Although relative luminescence output will vary with different instruments, this variation should not affect assay performance. Any solid white plates that fit your luminometer should be suitable. We don’t have specific recommendations but here are some examples:
- Corning® Costar plate (Cat.# 3917)
- Greiner Bio-One CELLSTAR plate (Cat.# 655073)
- Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated (Cat.# 136101/02)
- Falcon® 96-well Solid White Flat Bottom TC-treated (Cat.# 353296)
What instrument settings should I use for this assay?
Most standard plate readers designed for measuring luminescence are suitable for this assay. Some instruments do not require gain adjustment, while others may require optimization of the gain settings to achieve sensitivity and dynamic range. An integration time of 0.3–1 second per well should serve as guidance. For exact instrument settings, consult your instrument manual. Although relative luminescence output will vary with different instruments, this variation should not affect assay performance.
What experimental controls should I use?
- Perform the assay in parallel wells with and without the caspase-1 selective inhibitor, Ac-YVAD-CHO, to calculate the portion of activity due to caspase-1 activity specifically.
- For both measurements, include no-compound (vehicle only) control and no-cell background controls.
- Use a positive control known to induce caspase-1 activity with your sample type under your test conditions for comparison. See Technical Manual #TM456 for examples of inflammation inducers.
What are the main assay considerations?
- It is important that cells are as healthy as possible before testing for inflammasome activation. You may need to optimize cell number, dose, and the assay timing to detect active caspase-1.
- Measuring released caspase-1 activity in culture medium separately may provide a better signal-to-background ratio than measuring activity in cultured cells and medium together.
- Including the proteasome inhibitor, MG-132, in the reagent is important to eliminate nonspecific proteasome-mediated cleavage of the substrate. To confirm that MG-132 inhibition and Ac-YVAD-CHO inhibition are complete, we recommend that you measure luminescence for each plate at 60 minutes, 90 minutes and 120 minutes incubation.
Do you recommend any resources for background information?
For additional information, view the product page.
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