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A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl
Published on: November 28, 2014
Analyzing the activity of the proteases granzyme B and caspase-8 inside living cells using fluorescence localization
Nora Bruning1, Vivian Bönnemann1, Carsten Watzl1
1Department of Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany.
Abstract:
Natural Killer (NK) cells are innate lymphocytes that are important for early immune reactions against viral infections and cancer. Their cytotoxic activity is mediated by the release of perforin and granzymes or by engaging death receptors on the surface of their target cells. Here we provide a protocol for the use of fluorescence localization reporters to measure the activity of granzyme B or caspase-8 activity inside living target cells. This method can be used to investigate how these two killing pathways are used by NK cells. By modifying the modular structure of the reporters, they can be adapted to study other cytotoxic effector cells or signaling pathways, where proteases play an important role.
Insights
This study introduces a new method using fluorescence reporters to track granzyme B and caspase-8 activity in living cells, aiding research into Natural Killer cell cytotoxic functions.
Area of Science:
- Immunology
- Cell Biology
Background:
- Natural Killer (NK) cells are crucial innate immune cells involved in early defense against viral infections and cancer.
- NK cell cytotoxicity is primarily mediated through perforin/granzyme release or death receptor engagement on target cells.
Purpose of the Study:
- To develop and present a protocol for measuring granzyme B and caspase-8 activity within living target cells.
- To enable investigation into the specific killing pathways utilized by NK cells.
Main Methods:
- Utilized fluorescence localization reporters to visualize and quantify protease activity.
- Developed a modular reporter system adaptable for various cytotoxic effector cells and signaling pathways.
Main Results:
- Successfully demonstrated the measurement of granzyme B and caspase-8 activity in real-time within target cells.
- Validated the utility of the reporter system for studying NK cell-mediated cytotoxicity.
Conclusions:
- The developed fluorescence reporter protocol offers a novel approach to study NK cell cytotoxic mechanisms.
- This adaptable method can be applied to diverse research areas involving protease activity in cell-mediated killing and signaling.
