Process for measuring apoptosis in mammals

摘要

A method for measuring apoptosis in mammals by comparing the ratio of untransfected cells and apoptotic cells, is new. A process for measuring apoptosis comprises: (a) transfecting transient mammalian cells with: (i) a plasmid comprising a DNA encoding a polypeptide, which may have an apoptotic or anti-apoptotic effect; or (ii) a plasmid comprising a DNA sequence which is tested for modulation of its expression by the presence of apoptosis-inducing or -preventing substances; and (iii) a plasmid comprising DNA which encodes a fluorescent marker protein; (b) culturing the cells in a nutrient medium until the polypeptide and its effects on apoptosis are apparent; (c) harvesting and fixing the cells so that the fluorescent protein remains in the cells, while apoptosis-induced DNA fragments can diffuse out of the cells; (d) measuring the apoptotic cells by determining the DNA content; (e) measuring the amount of transfected cells by determining the cells comprising the fluorescent marker protein; and (f) determining the ratio of apoptosis in transfected cells by comparing the results of (d) and (e). An Independent claim is also included for a kit for carrying out the method above, comprising a number of containers, comprising: (a) components for cell transfection; (b) a plasmid encoding the fluorescent marker protein; (c) an empty plasmid for inserting DNA of interest as well as control measurements; (d) primary fixative; (e) secondary fixative or permeabilisation solution; (f) wash solutions; and (g) a DNA finding coloring agent.