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.
Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, Glasgow, G11 6NT, UK. amm43w@clinmed.gla.ac.uk
Angela M
AM
Grierson
Paul
P
Mitchell
Claire L
CL
Adams
Allan McI
AM
Mowat
James M
JM
Brewer
Margaret M
MM
Harnett
Paul
P
Garside
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