Multi-parametric phospho-flow cytometry: a crucial tool for T lymphocyte signaling studies.
Tools such as protein immunoblotting have proven benefits for investigating T lymphocyte signaling but have several drawbacks such as the number of cells required and the difficulty of distinguishing subset-specific differences without expensive and invasive cell sorting. Recent advances in immunology and the identification of T lymphocyte sub-populations making up only a very small fraction of the total population highlight the importance of studying signaling in those small subsets in a feasible, cost-effective, high-throughput manner. To this end, we have developed a simplified protocol to study both intracellular phosphorylation patterns of important signal transduction molecules concomitantly with T cell surface marker expression. A multi-parametric analysis may allow the quantification of the phosphorylation of up to five signaling molecules in CD4 and CD8 T lymphocytes and their naïve, central memory, effector memory, and TEMRA subsets. This enables precise identification of subset-specific signaling and alterations of signaling pathways in physiological and pathological situations. The importance of such detailed analysis is discussed.
Center for Medical Research ZMF, Tübingen Aging and Tumor Immunology group, Tübingen, Germany.
David
D
Goldeck
Ivy
I
Low
Nurhidaya Binte
NB
Shadan
Seri
S
Mustafah
Graham
G
Pawelec
Anis
A
Larbi
D000818:Animals; D015703:Antigens, CD / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000378:metabolism*
D015703:Antigens, CD; D000954:Antigens, Surface