Micromixing-stirring device for flow cytometry studies.

A special device able to perform fast mixing and stirring of samples with different reagents was placed inside the flow chamber of a flow cytometer. This mixing device is coupled with stepper-motor driven syringes which are able to inject desired volumes of sample and reagents under computer control. The fast mixing is performed by means of a small magnetic rod vibrating in alternating magnetic field. The device may work both in continuous and stepwise manner delivering defined volumes of sample and reagent at set time intervals. Internal volume of the mixing chamber is about 0.2 mu l. The frequency of stirring can vary up to 1 kHz thus providing different modes: mild mixing, breaking of cell aggregates, and cell-membrane rupture. This device was used for studies of DNA content distribution in cell cycle analysis, binding kinetics of DNA-specific dyes and for analysis of chromosome sets from prestained mitotic cells. It is shown that the breaking of aggregates is essential for correct estimation of the S-phase. The most significant effects are observed in the case of activated lymphocytes and for the analysis of samples from solid tumors. Breaking of cell aggregates has been achieved by a simplified device placed just prior to the flow chamber.