EGF cell surface receptor quantitation on ocular cells by an immunocytochemical flow cytometry technique.
A method is presented for the rapid flow cytometric determination of epidermal growth factor (EGF) receptor densities on the surface of cultured ocular cells. The technique uses a biotinylated monoclonal antibody directed against the EGF receptor in conjunction with a streptavidin-bound fluorochrome and requires the specific fluorescence per cell to be measured as a function of ligand and receptor concentration. Because the measurement is noninvasive and restricted to cell surface-bound material, the cells can be kept in a physiologic environment, even at the moment of assay. Calculated receptor densities ranged from 5142/cell (infant human corneal endothelium) to 35,678/cell (infant human keratocytes) to greater than 5 x 10(5)/cell for an A431 control cell line. Species and donor age differences were noted, as was transient receptor downregulation after EGF administration. Flow cytometry represents a valuable time saving procedure for large scale applications while providing the same level of sensitivity as standard radioimmunoassays. This technique is applicable to quantitation of other growth factor cell surface receptors and could greatly expand the use of flow cytometry in the research laboratory.
Lopez
J G
JG
Louisiana State University Medical Center School of Medicine, New Orleans 70112-2234.
Chew
S J
SJ
Thompson
H W
HW
Malter
J S
JS
Insler
M S
MS
Beuerman
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D003315:Cornea / Q000737:chemistry*; D066246:ErbB Receptors / Q000032:analysis*; D005347:Fibroblasts / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007223:Infant; D008875:Middle Aged; D011817:Rabbits; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D066246:ErbB Receptors