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Immunomagnetic separation reagents as markers in electron microscopy
Phyllis J Fisher1, Margaret J Springett, Allan B Dietz
1Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First St., S.W., Rochester, MN 55905, USA. fisher.phyllis@mayo.edu
Antibodies coupled to magnetic particles have been employed for immunomagnetic cell isolation, but their consequent use for electron microscopy (EM) has not been evaluated. We used commercial antibodies coupled to iron-dextran to isolate T cells and monocytes/macrophages by immunomagnetic adsorption from normal human peripheral blood mononuclear cells. Subsequently, we studied the association of electron-dense immunomagnetic reagents with cell membranes. CD14-positive monocytes/macrophages isolated from fixed peripheral blood mononuclear cells retained electron-dense beads on the plasma membrane, while live cells internalized them. Flow cytometry and electron microscopy measurements of the percentage of cells that bound a CD4-specific immunomagnetic reagent in pan-T cell isolates (containing numerous T cell subtypes) were indistinguishable. The immunomagnetic reagent associated with cells could be secondarily labeled by secondary antibody coupled to colloidal gold. This study shows that these reagents used for cell isolation or just labeling, remain associated with their targets at the cell membrane. Immunomagnetic reagents allow "capturing" of rare cells from complex mixtures, purifying and concentrating them in a single step for subsequent electron microscopy. The large number of commercially available immunomagnetic reagents specific for different human, mouse and rat antigens provides additional resources for visualization of cellular ultrastructure.
Antibodies coupled to magnetic particles have been employed for immunomagnetic cell isolation, but their consequent use for electron microscopy (EM) has not been evaluated. We used commercial antibodies coupled to iron-dextran to isolate T cells and monocytes/macrophages by immunomagnetic adsorption from normal human peripheral blood mononuclear cells. Subsequently, we studied the association of electron-dense immunomagnetic reagents with cell membranes. CD14-positive monocytes/macrophages isolated from fixed peripheral blood mononuclear cells retained electron-dense beads on the plasma membrane, while live cells internalized them. Flow cytometry and electron microscopy measurements of the percentage of cells that bound a CD4-specific immunomagnetic reagent in pan-T cell isolates (containing numerous T cell subtypes) were indistinguishable. The immunomagnetic reagent associated with cells could be secondarily labeled by secondary antibody coupled to colloidal gold. This study shows that these reagents used for cell isolation or just labeling, remain associated with their targets at the cell membrane. Immunomagnetic reagents allow "capturing" of rare cells from complex mixtures, purifying and concentrating them in a single step for subsequent electron microscopy. The large number of commercially available immunomagnetic reagents specific for different human, mouse and rat antigens provides additional resources for visualization of cellular ultrastructure.