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The Caspase-Glo® 1 Inflammasome Assay is a simple, homogeneous, bioluminescent method to selectively measure the activity of caspase-1, a member of the cysteine aspartic acid-specific protease (caspase) family and an essential component of the inflammasome.
Inflammasomes are protein complexes induced by diverse inflammatory stimuli. Innate immune cells respond to pathogens and other danger signals with inflammasome formation and conversion of procaspase-1 zymogen into catalytically active caspase-1. Caspase-1 activation results in the processing and release of cytokines IL-1β and IL-18, as well as pyroptosis, an immunogenic form of cell death. The Caspase-Glo 1 assay is sensitive enough to measure caspase-1 activity directly in cells or in medium in multiwell plates.
Following caspase cleavage of the Z-WEHD subsrate (Z-WEHD-aminoluciferin), a substrate for luciferase (aminoluciferin) is released, resulting in luciferase activity and generation of light by a proprietary, thermostable, recombinant luciferase.
The assay’s easy and flexible workflow requires no sample preparation or manipulation, simply add, mix and then measure luminescence after only one hour. The assay takes significantly less time and involves fewer steps compared to conventional Western blot and ELISA analyses.
The selective caspase-1 substrate (Z-WEHD) enables detection of catalytically active caspase-1 in cells or culture media and quantitative measurement of inflammasome activity. Inclusion of the proteasome inhibitor MG-132 in the reagent eliminates non-specific proteasome substrate cleavage, enabling sensitive detection of caspase-1. The assay includes a caspase-1 specific inhibitor (Ac-YVAD-CHO) to confirm specific activity in parallel samples and distinguish caspase-1 activity from other caspases.
Inflammation and apoptosis caspases at equimolar concentrations were tested with the assay, +/- Ac-YVAD-CHO, and caspase activity was measured after 20 minutes. Ac-YVAD-CHO at 1 µM inhibited 99% of the caspase-1 activity, whereas it had minimal effect on the other cross-reacting caspases (5,3 and 6)
THP-1 cells were grown in RPMI 1640 medium and added to plates at 5 x105 cells/ml. After differentiation with PMA in 96-well plates they were treated with flagellin (1 μg/ml for one hour) or nigericin (20 μM for two hours). Then 50 µl of Caspase-Glo® 1 Reagent or Caspase-Glo® 1 YVAD-CHO Reagent was added to each well. Luminescence was recorded after 30 minutes.
Caspase-Glo® 1 Inflammasome Assay monitors caspase-1 activity in peripheral blood mononuclear cell (PBMC) supernatant. Cells were stimulated with or without LPS then subsequently stimulated with ATP for 30 minutes. Cells were then pelleted and supernatant was assayed for caspase-1 activity.
While caspase-1 activity can be directly measured in cells in microwell plates, assay of transferred medium measures released caspase-1 activity, leaving cells intact to assess other parameters of cell health.
Caspase-Glo 1 Inflammasome Assay Multiplexed with Cell Viability and Cell Death Assays. THP-1 cells were grown in RPMI 1640 medium supplemented with 10% FBS in a 37°C incubator with 5% CO2. Cells were added to plates at 5 x 105 cells/ml in 100 µl of medium and differentiated with PMA (20 nM, 3 days) in 96-well plates followed by treatment with flagellin (1 µg/ml, 1 hour) or nigericin (20 µM, 2 hours). In Panel A CellTox™ Green Reagent was added to the cells, and fluorescence was recorded after 90 minutes. In Panel B cell viability was monitored with CellTiter-Glo® or RealTime-Glo™ MT Cell Viability Assay. Luminescence was recorded at 10 minutes and 90 minutes, respectively.
Caspase-Glo® 1 Inflammasome Assay
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Caspase-Glo® 1 Buffer
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U.S. Pat. Nos. 7,148,030, 7,384,758, 7,666,987 and 8,071,328 and other patents and patents pending.
U.S. Pat. Nos. 6,602,677, 7,241,584, 8,030,017 and 8,822,170 and other patents and patents pending.
A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.
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