Figure 2

COS1(B3GALNT1) cells transfected with M_GBGT1(AlaGlyGly) or H_ABO-A(AlaGlyGly) expressed FORS1 antigen. Cells transfected with selected expression constructs were analyzed for FORS1 glycan expression by detecting AF488 fluorescence at 530 nm, together with the expression of mRFP fluorescence at 575 nm. On the top row, the first two figures correspond to the transfection experiments of the original M_GBGT1 construct. Single viable cell population was selected based on forward (FSC-A) and side (SSC-A) scattering amplitudes on the left. Events were plotted in pseudocolor with red representing higher frequency and dark blue the lowest, and the gate set to select cell population for analysis is indicated. The same selection was maintained for all the other samples. Next to it, a contour plot shows the mRFP fluorescence intensity, used as transfection control, on the y-axis, and the AF488 fluorescence corresponding to FORS1 on the x-axis. Both axes are logarithmic in scale. The upper red rectangle represents the gate containing the mRFP positive cells and the percentage of mRFP + cells is also indicated at the upper left corner. The green rectangle corresponds to the gate containing the AF488-positive cells, and the percentage is indicated at the lower right corner. The other graphs show the results from flow cytometry of cells transfected with other constructs.