Abstract
FLOW cytometry enables assessment of the relative concentrations of proteins on the cell surface or within the cell with single-cell resolution. Many molecules of cell signaling, however, are expressed at low levels, perhaps at hundreds to only a few thousands of copies per cell. Being below or near the edge of resolution, detection of these targets using conventional flow cytometers and staining techniques is not reliable. Flow cytometric instrument detection limits in the fluorescein channel, for example, range from 1,000–3,000 molecules per cell. Cellular autofluorescence also plagues measurement sensitivity: the 98th percentile of autofluorescence in various primary cell populations is in the range of 2,500–4,000 fluorescein molecules. Detection sensitivity may be increased by using sandwich techniques, quantum dots or far red emitting dyes, among others.
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