( to the use of antigensthat are expr...)Detection of antibody-stained cell,,,surface and intracellular protein targets,,,with the Agilent 2100 bioanalyzer

to the use of antigens that are expressed on the cell surface. Following fixation and permeabilization of cells, intracellular antigens can be easily detected by antibody staining. Detection of proteins generally depends on the availability of a suitable antibody, many of which are commercially available in a fluorescently conjugated form. As the Agilent 2100 bioanalyzer has the highest sensitivity in the red, with an excitation wavelength of 635 nm and a detection wavelength of 685 nm, APC, or Cy5 can be used as dyes. Counterstaining the cells with the live cell dye calcein allows the discrimination between live and dead antibody stained cells. In contrast, the measurement of intracellular proteins requires permeabilization and fixation of cells prior to antibody labeling. Therefore, staining with Calcein is not advisable. In this case cells can be counterstained with the nucleic acid-specific-dye SYTO16.
Staining of cell surface proteins with antibodies may introduce artifacts that should be eliminated by special experimental procedures and by carefully and critically examining the results obtained. On the one hand, crosslinking of several receptor molecules by bivalent antibodies may lead to receptor internalization, where the receptor becomes internalized together with the bound antibodies as described for endothelial growth factor (EGF)-Receptor 1. On the other hand, the extracellular part of the receptor molecule may be shed from the cell surface, as described for CD442, 3. As these effects require membrane alterations, they are prevented if the cells are fixed or kept continuously on ice and in a buffer containing low amounts of sodium azide.
Experimental
The Agilent 2100 bioanalyzer and Cell Assay Extension were obtained from Agilent Technologies Deutschland GmbH (Waldbronn, Germany)
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