Inflammasomes are protein complexes induced by a diverse set of inflammatory stimuli. Innate immune cells respond to pathogens and other danger signals with the formation of various inflammasomes and the activation of caspase-1. Caspase-1 activation leads to processing and release of the cytokines, IL-1β and IL-18, and an immunogenic form of programmed cell death called pyroptosis.
While the innate immune system has evolved as a rapid defense mechanism to infection, and inflammation is a protective response to infection, dysregulation can lead to chronic inflammatory diseases. Chronic inflammation is linked to many diseases including asthma, rheumatoid arthritis, inflammatory bowel syndrome, Alzheimer’s, Type II diabetes and heart disease. As more attention is focused on the role of inflammation in human disease, understanding the role of the inflammasome and having the ability to measure inflammasome activation will become increasingly valuable.
Challenges of Measuring Inflammasome Activation
Inflammasome activation is typically measured by Western Blot or ELISA. While these are standard methods, both are cumbersome and time consuming and may require making lysates or using serum-free supernatant. Other drawbacks include variable caspase-1 antibody quality and the limited dynamic range of ELISAs. More importantly, Western blots and ELISAs do not necessarily monitor active enzyme. Inflammasome activity can be missed using a Western blot because oligomerization but not cleavage of caspase-1 is required for activity
ELISA analysis of released IL-1β as a marker for inflammasome activation can be complicated by IL-1β that may be processed extracellularly by other proteases
or antibody cross-reactivity with pro-IL-1β that can be released when there is cytotoxicity.
A Better Alternative
Ease of Use
The Caspase-Glo® 1 Inflammasome Assay is a better alternative to conventional methods of detecting inflammasome activity. The simple “add-mix-measure” protocol eliminates cell washing, medium removal and multiple pipetting steps that are required for Western blot and ELISA analysis. The Caspase-Glo® 1 Inflammasome Assay can directly measure caspase-1 activity in cells or medium from cultured cells in microwell plates. By measuring released, active caspase-1 from transferred culture medium, you can preserve the biological sample for downstream analysis with other assays.
Figure 1. Caspase-Glo® 1 Inflammasome Assay Workflow
The Caspase-Glo® 1 Inflammasome Assay assesses inflammasome activation by directly monitoring and selectively measuring activated caspase-1. The Caspase-Glo® 1 Reagent contains Z-WEHD-aminoluciferin, a selective substrate that is sensitive enough to detect caspase-1 activity in cells. Caspase-1 cleaves the substrate releasing aminoluciferin, a substrate for luciferase, resulting in the luciferase reaction and light production.
The addition of MG-132 inhibits non-specific proteasome activity, enabling direct detection of caspase-1 activity. Since the assay detects only catalytically active caspase-1, you can determine the precise time course of inflammasome activation and deactivation. The assay can also differentiate inflammasome activation and the resulting pyroptosis from apoptosis or necrosis (Figure 4). Using the Ac-YVAD-CHO caspase-1 inhibitor in parallel samples confirms the activity is caspase-1 specific. Ac-YVAD-CHO inhibits all of the caspase-1 signal but does not inhibit other cross-reacting caspases, such as caspases 3 and 6 which are both associated with apoptosis. Therefore, a signal inhibited by YVAD-CHO indicates caspase-1 activity, whereas a signal that is not inhibited by YVAD-CHO indicates another caspase.
In-well Detection & Multiplexing
The sensitivity and low background of the assay enable it to be used on cells in culture in microwell plates or with cell culture supernatant. In addition, you can perform same-well multiplexing with other compatible assays (e.g., CellTox™ Green Cytotoxicity Assay). Multiplexing will allow you to save time and distinguish between different outcomes of caspase-1 activation by monitoring cell death and cytokine release. Detection of caspase-1 activation along with cell death indicates pyroptosis. You can also multiplex this assay with an IL-1β ELISA by removing a portion of the culture medium and measuring released IL-1β followed by the caspase-1 assay on the remaining cells and culture medium.
Other Key Benefits and Features
The Caspase-Glo® 1 Inflammasome Assay is ideal for automated high-throughput screening. The homogeneous protocol enables easy scaling and automation in 96- and 384-well formats. The high throughput capability of this convenient caspase-1 assay will allow you to screen for modulators of inflammasome activation. Although luminescence can be measured after only one hour, the luminescent caspase-1 signal is stable in the Caspase-Glo® 1 Reagent (half-life >3 hours). This allows you to read plates over a few hours and eliminates the need for a luminometer with reagent injectors.
Measuring inflammasome activation provides valuable information for understanding many diseases associated with inflammation. Current approaches for monitoring inflammasome activation can be arduous and lack specificity. The Caspase-Glo® 1 Inflammasome Assay provides a simple and direct method for in-well detection of inflammasome activation that offers greater sensitivity and flexibility than traditional methods.