Dissociation and purifying of the retinal pigment epithelial cells by gradient centrifugation / 中华眼底病杂志

ABSTRACT

Objective To investigate the effect of dissociation of the human retinal pigment epithelium (RPE) cells by ficoll hypaque gradient centrifugation. Methods The primary human RPE cells and subcultured human RPE cells were dissociated with ficoll gradient centrifugation solution (d= 1.077 g/ml) and the same divided cells as the control were dissociated with routine normal culture medium centrifugation. The Trypan blue (0.4%) rejection staining was used, and the mouse anti-human monoclonal antibody and fluorescein isothiocyanate (FITC) labeled rabbit anti-mouse IgG were utilized for indirect immunoreactivity for the test of human cytokeratin (CK) in active RPE cells cytoplasm. Flow cytometry assay was used to analyzed the percentages of CK positive staining RPE cells. simultaneously, the cells configuration, growth condition, the rate of clone formation, and the purifying result were observed under the fluorescent and confocal microscope. Results The survival rate and positive rate of CK of RPE cells in experimental group were higher than those in the control ( P