Direct quantitation of T cell signaling by laser scanning cytometry.
Current flow cytometric technology allows quantitative assessment of surface and intracellularly expressed molecules on isolated cells. However, the need to disrupt tissues prevents correlation of phenotypic expression with anatomical location. In contrast, immunohistochemistry in conjunction with conventional or confocal microscopy allows localisation of staining, but little in the way of quantitation. The laser scanning cytometer (LSC) allows a combination of both approaches, as it can apply quantitative flow cytometric laser technology to intact tissue. The purpose of this protocol is to describe in vitro and ex vivo methods for quantifying cell signaling molecule expression and activation within antigen-specific T cells by laser scanning cytometry.
Grierson
Angela M
AM
Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, Glasgow, G11 6NT, UK. amm43w@clinmed.gla.ac.uk
Mitchell
Paul
P
Adams
Claire L
CL
Mowat
Allan McI
AM
Brewer
James M
JM
Harnett
Margaret M
MM
Garside
Paul
P
D000818:Animals; D000941:Antigens / Q000276:immunology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D047569:Laser Scanning Cytometry / Q000379:methods*; D008198:Lymph Nodes / Q000201:enzymology; D051379:Mice; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
D000941:Antigens; D048049:Extracellular Signal-Regulated MAP Kinases