Green fluorescent protein (GFP) is a particularly important reporter gene used in various transformation studies. Expression of GFP fluorescence can be visually monitored under UV or blue excitation in transformed cells. However, quantifications of fluorescence expression using fluorimetric methods are limited to average expression in tissues and cannot be assessed in single cells. An improved protocol to determine quantitative single cell fluorescence was developed using GFP-transformed tobacco leaf protoplasts measured by multiparameter flow cytometry. It was shown that a Percoll density gradient or sucrose flotation are essential for optimal separation. Fluorescent protoplasts and those expressing only background autofluorescence were successfully separated using three-parameter analysis. For clustered subpopulations, relative fluorescence intensity and proportions of cells with expressed or non-expressed fluorescence can be measured. Further applications of this novel procedure are discussed.
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