A method for detecting intracellular perforin in mouse lymphocytes.
J Immunol
; 193(11): 5744-50, 2014 Dec 01.
Article
in En
| MEDLINE
| ID: mdl-25348626
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
T-Lymphocytes, Cytotoxic
/
Intracellular Space
/
Granzymes
/
Perforin
/
Immunological Synapses
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
J Immunol
Year:
2014
Type:
Article