Fig. 4. Effect of Cu²⁺ on the cell-surface distribution of PrP^C. SH-SY5Y cells stably expressing PrP^C were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence or presence of 100 µM Cu²⁺ along with 500 µM tyrphostin A23. Where indicated, cells were incubated at 4°C for 10 minutes with PBS containing 1% Triton X-100 prior to fixation. Cells were fixed, incubated with Alexa488-conjugated secondary antibody and viewed using a DeltaVision Optical Restoration Microscopy System. (A) Images were taken of ten Z-slices from the top of the cell, and of an individual Z-slice from the middle of the cell. Bars, 10 µm. (B) Cell-surface distribution of PrP was measured using ImageJ as described in the Materials and Methods. Average fluorescent intensities per unit length of membrane were as follows: –Cu²⁺, 15.14 units; –Cu²⁺ + Triton X-100, 13.46 units; +Cu²⁺, 16.22 units; +Cu²⁺ + Triton X-100, 8.58 units. (C) The percentage of the cell surface stained for PrP^C was determined from an individual Z-slice image in panel (A). Results are the mean±s.e.m. of three individual experiments, in each of which 30 cells were measured. Statistical differences, using Student's t-test (n=90), with probability values of P<0.05 were taken as significant.