Detection and isolation of lectin-transfected COS cells based on cell adhesion to immobilized glycosphingolipids.
Two methods are described, one for detection and one for isolation of COS cells transiently expressing vertebrate lectins. The methods are based on specific cell adhesion to polystyrene microwells or magnetic beads adsorbed with glycosphingolipids. In the first method, glycolipids were adsorbed to wells of 96-well polystyrene plates. A suspension of lectin-transfected COS cells was added and the plate was incubated to allow cell adhesion to occur. The plate was then immersed in buffer, inverted (while immersed), and placed in a fluid-filled Plexiglas centrifugation chamber which was sealed to avoid introducing an air-liquid interface. The chamber, with the inverted plate enclosed, was centrifuged to remove nonadherent cells. The plate was then removed from the carrier (while immersed) and righted, and adherent cells were quantitated enzymatically or immunochemically using a 96-well plate reader. COS cells transfected with an expression plasmid carrying the gene for the rat Kupffer cell lectin (fucose and N-acetylgalactosamine specific) adhered specifically to globotetraosylceramide. Glycolipid- and lectin-specific cell adhesion was readily detected even when COS cells were transfected with a plasmid mixture containing 0.5% lectin-carrying plasmid and 99.5% irrelevant plasmid. This sensitivity will facilitate screening of plasmid pools to detect and isolate plasmids expressing mammalian lectin genes. To isolate COS cells transiently expressing lectin, glycosphingolipids were adsorbed to carboxylated magnetic polystyrene microspheres, which were mixed with the lectin-transfected COS cells. Adherent cells were collected on a fixed magnet and plasmid recovered for subsequent amplification.
Yang
L J
LJ
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland, 21205, USA.
Zeller
C B
CB
Schnaar
R L
RL
D000327:Adsorption; D000818:Animals; D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D006028:Glycosphingolipids / Q000378:metabolism; D007728:Kupffer Cells / Q000737:chemistry; D037102:Lectins; D008280:Magnetics; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D006028:Glycosphingolipids; D037102:Lectins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C039707:galactose receptor