Methods of successive multiparametric cytochemistry and microfluorometry on identical cells with special reference to...

The study of cell cycle kinetics in relation to regulatory mechanisms in embryonic development is considered quite important, but many technical difficulties still remain owing to the complexity of embryonic systems. To facilitate such study, a novel method of multiparametric microfluorometry was developed and is presented here. In many other multiparametric methods, various cytochemistries and fluorometries are conducted at the same time, whereas in our system only one type of cytochemistry and its microfluorometry are performed first and then other types subsequently. Our system is composed of two parts, a method for combining various types of quantitative cytochemistries to be performed in succession on identical cells and a method that permits the implementation of cytochemistries and microfluorometries in succession using in combination a microfluorometer, TV camera, video recorder, digital Telopper, and microcomputers. A method for the evaluation and normalization of microfluorometric data on DNA content is described. The cell cycle phases of each cell can be accurately distinguished on the basis of DNA content and BrdU uptake, using a statistical method. A cell cycle traverse study using embryonic avian scale dermal cells was carried out in which the four parameters of DNA content, double labeling with BrdU and [3H]AdR, and time lapse between the two labelings were included. As an example of its application to molecular cytochemistry, in situ nick translation was conducted on developing scale dermal cells. The special features and scope of application of the present system are discussed.