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Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
Published on: December 17, 2013
Measurement of polyclonal and antigen-specific cytotoxic T cell function
W E Biddison1, R Lichtenfels, M Adibzadeh
1National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.
Current Protocols in Immunology
|April 25, 2008
Summary
This study details two methods for measuring T cell cytotoxic function: one using anti-CD3 antibody for polyclonal stimulation and another for antigen-specific responses. These assays, including the calcein release assay (CARE-LASS), are adaptable for various immune cells.
Area of Science:
- Immunology
- Cellular Immunology
- Cytotoxicity Assays
Background:
- Measuring in vitro cytotoxic function of human T cells is crucial for immunological studies.
- Cytolytic effector cells can be stimulated either polyclonally using anti-CD3 antibody or specifically with an antigen.
Purpose of the Study:
- To describe two distinct assay protocols for measuring T cell-mediated cytotoxicity.
- To present methods for both anti-CD3-mediated and antigen-specific cytotoxicity.
- To adapt the calcein release assay (CARE-LASS) for various immune cell types and stimulation methods.
Main Methods:
- Development of two sets of assays for cytolytic T cell function.
- Assay 1: Measuring anti-CD3-mediated cytotoxicity via polyclonal T cell stimulation.
- Assay 2: Measuring antigen-specific cytotoxicity using specific stimulating antigens.
- Adaptation of the calcein release assay (CARE-LASS) for antigen-activated cytotoxic T lymphocytes (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells.
Main Results:
- Established protocols for quantifying T cell cytotoxic function through both polyclonal and antigen-specific stimulation.
- Demonstrated the utility of the calcein release assay (CARE-LASS) for assessing cytotoxicity in multiple immune cell populations.
- Outlined minor protocol modifications for adapting assays to polyclonal T cell activation with calcein-labeled target cells.
Conclusions:
- Two reliable methods are presented for measuring in vitro T cell cytotoxic function.
- The calcein release assay (CARE-LASS) is a versatile tool for assessing cytotoxicity across different immune cell types and stimulation conditions.
- These assays provide valuable tools for immunological research and the study of cellular immune responses.
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