Polysialylation of cell-surface proteins on live PC12 cells.

<p>PC12 cells were incubated with the three PSTs in the absence and presence of 5 mM CMP-Neu5Ac for 1 hour at 37°C. Cell-surface PSA was detected using PSA-NCAM immunocytochemistry and nuclei were detected with Hoechst 33258 and confocal microscopy imaging. Following treatment of PC12 cells with all three PSTs in the presence of CMP-Neu5Ac specific cell-surface PSA labeling was detected (as indicated in the panels denoted “+CMP-Neu5Ac). Control reactions were also performed with the CMP-Neu5Ac absent; in these reactions PSA labeling was not detected. Quantitation of cellular polysialylation was performed using ImageJ (v 10.2) on the immunocytochemical images from two independent experiments by measuring the fluorescent intensity of 15 randomly selected cells per image. Images were pre-processed by performing background subtraction (50 pixel rolling ball radius) prior to measuring pixel intensity. Relative fluorescence values were calculated by dividing the PSA intensity (green fluorescence) by DAPI intensity (blue fluorescence). Data were normalized to average+CMP fluorescence for each experiment (n = 2).