Spectrofluorometric assay for the quantitation of cell-tissue electrofusion.
Fluorometric assay for quantitating electrofusion, FAQE, was developed to measure the number of cells fused to intact tissue. The fluorescent vital dye hydroethidine is used in this method. The fluorescent intensity detected in the cell tissue hybrids is proportional to the number of individual cells fused. The number of cells fused was determined after fusion by lysing the epithelial layer with 0.2% sodium dodecyl sulfate and the supernatant fluids were measured in a spectrofluorometer and compared to established standard curves. The mean number of cells fused, in five separate experiments, was determined to be approximately 5000. All the experimental corneas had approximately the same number of fused cells with less than 10% variation. In addition, the technique was used to demonstrate an increase in the number of cells fused when multiple fusions were applied to the cell-tissue electrofusion system. These results demonstrate that FAQE can be utilized to quantitatively analyze the fusion yields.
Heller
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Department of Surgery, College of Medicine, University of South Florida, Tampa 33612.
D000818:Animals; D002459:Cell Fusion; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014407:Tumor Cells, Cultured