A method for detecting intracellular perforin in mouse lymphocytes.
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| Abstract |
Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.
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| Authors | Amelia J Brennan, Imran G House, Jane Oliaro, Kelly M Ramsbottom, Magdalena Hagn, Hideo Yagita, Joseph A Trapani, Ilia Voskoboinik |
| Journal | Journal of immunology (Baltimore, Md. : 1950) (J Immunol) Vol. 193 Issue 11 Pg. 5744-50 (Dec 01 2014) ISSN: 1550-6606 [Electronic] United States |
| PMID | 25348626 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
| Copyright | Copyright © 2014 by The American Association of Immunologists, Inc. |
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