Flow cytometric detection of the Golgi apparatus using antibodies to glycosyltransferases.
Intracellular glycosyltransferase protein expression can be assessed by flow cytometry. We report the detectability of the Golgi associated beta1,4 galactosyltransferase (GT) and alpha2,6 sialyltransferase (ST) upon permeabilization in Jurkat and EBV-JY cells representing a T- and B-lymphoid cell line, respectively. The method employs fixation with paraformaldehyde and permeabilization with saponin. It ensures reliable internalization of the antibody and little background staining and does not cause leakage of the antigen. We first applied monoclonal antibodies to GT for establishment of this method by flow cytometry. The obtained flow cytometric signal could be localized to the Golgi apparatus by confocal laser scanning microscopy. To exclude interference from possible cell-surface staining, measurements were carried out on non-permeabilized cells. No signal was found in Jurkat cells, while low but measurable ecto-galactosyltransferase was found on EBV-JY cells. F(ab)'2 fragments of polyclonal antisera to GT and ST were generated and shown to be useful for double indirect staining with the monoclonal antibody. The method described here permits relative assessment of Golgi glycosyltransferase expression in lymphoid cells by flow cytometry.
Mrkoci Felner
K
K
Institute of Physiology, University of Zürich, Switzerland.
Niederer
E
E
Berger
E G
EG
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005700:Galactosyltransferases / Q000276:immunology* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D019169:Jurkat Cells; D008214:Lymphocytes / Q000648:ultrastructure*; D051379:Mice; D018613:Microscopy, Confocal; D012799:Sialyltransferases / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D005700:Galactosyltransferases; D012799:Sialyltransferases