Isolation of the phagocytic compartment from macrophages using a paramagnetic, particulate ligand.
Isolation of Fc receptor-dependent phagosomes from a macrophage cell line, J774 A.1, was accomplished using antibody-opsonized, paramagnetic beads. Following binding and ingestion of these beads, cells were homogenized in a standard membrane isolation buffer. Phagosomes containing the trapped paramagnetic beads were isolated by subjecting the whole cell homogenate to a magnetic field. The method is extremely simple and the preparation of an enriched phagosome fraction from a whole cell homogenate is rapid and highly selective. The method should provide useful starting material for investigators interested in cytoskeletal involvement in phagocytosis, in kinetic studies of ingestion, in phagosome-lysosome fusion, and in the ability of a particular ligand to initiate phagocytosis.
Lutz
D A
DA
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute, University of Louisville School of Medicine 40292.
Chen
X M
XM
McLaughlin
B J
BJ
D015720:5'-Nucleotidase / Q000032:analysis / Q000378:metabolism; D000135:Acid Phosphatase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008264:Macrophages / Q000502:physiology* / Q000648:ultrastructure; D008280:Magnetics; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D008970:Molecular Weight; D015388:Organelles / Q000502:physiology / Q000648:ultrastructure*; D010587:Phagocytosis; D011961:Receptors, Fc / Q000378:metabolism
D015415:Biomarkers; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D011961:Receptors, Fc; D000135:Acid Phosphatase; D015720:5'-Nucleotidase