Figure 1. Schematics of the glycosphingolipids produced by L-M(TK-) cells and the methodology used.

A. Biosynthetic pathways from lactosylceramide to SSEA-3 and Forssman antigens. The monosaccharides are color and shape coded as the legend details. UDP is uridine diphosphate and Cer is ceramide. Glycosyltransferase activities are shown on top of the arrows and red dashed lines indicate the epitopes recognized by the antibodies used. The right legend describes the abbreviation used throughout the article, the enzyme names and, between parentheses, the gene symbols. Glycosidic bonds are represented in a compact form (i.e. β4 standing for β 1→4). B. Flow chart and diagram explaining the protocol. Briefly, retroviral constructs containing glycosyltransferase cDNAs, an IRES and a fluorescent protein are transfected into packaging cells. The resulting viral particles are used to infect L-M(TK-) cells. After expansion, cells were detached from culture dishes. In the case of SSEA-3 expressing cells, cells were infected sequentially with all three viral particles (A4-GFP, B1-BFP and B5-RFP) and then triple positive cells were FACS sorted as shown schematically in the diagram. For Forssman antigen expressing cells, after infection with A4-GFP and B1-BFP viral particles, the double positive cells were FACS sorted and expanded first. Then they were infected with FS-RFP viral particles and finally the triple positive cells were isolated after a second FACS procedure as detailed in the flow chart.