Abstract
Publisher Summary This chapter describes a novel method that allows discrimination between intracellular and plasma membrane antigens and their quantitative measurement. It illustrates the procedure for studying human neutrophils along with advantages, limitations, and potential technical obstacles of this approach. The new flow-cytometric approach measures total cellular antigen and plasma membrane (exposed) antigen. Because flow cytometry measures each cell individually, subpopulations of cells with distinct antigen distributions are easily identified, and errors because of the calculation of average antigen content are eliminated. A further advantage of this method is that aliquots of the same samples used for flow cytometry may be inspected by fluorescence microscopy, thus permitting morphological examination of the intracellular antigen distribution as well as correlation with the quantitative results. The major requirements of this strategy are that the molecule studied must be immunoreactive, accessible to antibody, and not extracted or excessively altered by fixation and permeabilization. The new approach analyzes trafficking of the complement C3b/C4b receptor (CR1) of human neutrophils. As the flow cytometer measures all cell-associated fluorescence, it is essential that all of the immune reagents should be highly purified. Thus, affinity-purified specific antibodies are preferable to whole antisera or immunoglobulin fractions.
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