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RelevantRBC membrane proteins are selected based on the literature and these data are
compared to Mass Spectrometry (MS) measurements and the data in the erythrocyte
database. For Flow Cytometry (FACS) measurements, RBC ghosts (hemoglobin
depleted erythrocyte membranes) are prepared from a small drop of blood and
these ghosts are labeled by a fluorescent lectin derivative (e.g. wheat germ
agglutinin-AlexaFluor647, WGA-A647) to allow selective gating. Thereafter, the
ghosts are labeled with specific monoclonal antibodies, then with suitable
fluorescent secondary antibodies (for details see. The measurements are carried
out in a flow cytometer, preferably equipped with automatic high throughput
plate sampler.
Inorder to obtain quantitative results in the FACS assay, each membrane protein
and each selective first antibody has to be carefully titrated to give maximum
level interactions. Still, the results of the FACS measurements do not provide
an actual number of the given membrane protein per RBC, and data have to be
evaluated based on the protein levels obtained in a certain population or under
specific disease conditions.
