Determination of the mitotic index by microinjection of fluorescently labelled tubulin

Abstract

The microneedle injection technique is one of the most established procedures for the introduction of proteins into living cells. To analyse injected proteins which are important in cell cycle progression it is often necessary to determine the mitotic index. Measuring the mitotic index after microinjection is complicated because only a limited number of cells of the whole cell population is microinjected. Therefore, we attempted to establish a new method to determine the mitotic index using microinjection of fluorescently labelled α/β-tubulin into mammalian cells which allows to monitor the injected cells simultaneously with the determination of the mitotic index. We demonstrated that fluorescently labelled tubulin incorporates efficiently into the mitotic spindle apparatus. Fluorescence remains stable for several hours which is sufficient to observe the progression of cells through the M-phase of the cell cycle. The determination of the mitotic index with the method presented here gave similar results to those determined using other methods. With this method also different stages of mitosis can be visualized by analysing various steps of spindle formation. Thus, this rapid method allows the monitoring of the injected cells after microneedle injection and simultaneously the determination of the mitotic index.