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A special device able to perform fast mixing of samples with different reagents was placed inside the flow chamber of an ATC 3000 flow cytometer. This mixing device is coupled with a stepper motor driven syringe which is 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 an alterning magnetic field. The device works in a stepwise manner delivering defined volumes of sample and at set time intervals: The delay between sample injection can be varied from 1 sec up to 999 sec. Interval volume of the mixing and the chamber is about 0,2 µl. The frequency of stirring can vary up to l kHz thus providing different modes: mild mixing, cell aggregate breaking and cell membrane rupture.
Binding kinetics of DNA specific dyes (propidium, olivomycin, chromomycin) was also studied in the time range from 1 sec up to 15 min. In all cases at least 2 binding rates were found, fast binding, reflecting interaction of the DNA - ligands with free binding sites and a slower component that depends on the rearrangement from chromoatin structure. Time constants and amplitudes of both components were studied at different ionic strengths and ligand concentrations as well as after different cell fixation procedures. Mouse spleenocytes were used as a standard object for these experiments. This approach makes it possible to determine the experimental conditions for chromoatin staining and to reveal the functional differences between cells with equal DNA content.
This device was used for analysis of human chromosome sets. A new method for cell preparation and intracellular staining of chromosomes was developed. The method includes enzyme treatment, incubation with saponin and separation of prestained cells from debris on a sucrose gradient. Mitotic cells are injected one by one into the mixing/stirring device where they are ruptured, thereby releasing chromosomes which flow as a set to the point of analysis. Time gated accumulation of data in list mode files makes it possible to resolve chromosome sets arising from single cells. Data analysis algorithms were developed which match chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of a certain type.
Numerous experiments with flow analysis of human chromosome sets from single mitotic cells revealed the majority of metaphase cells give "single cell flow karyotypes" with clumped chromosomes. The percent of clumps is relatively constant despite particular protocols used for chromosome prestaining and release. We assumed that heteroclumping of chromosomes reflects the simple fact: final chromosome disjunction needs mechanical tensions produced by the anaphase spindle. Mechanical tension shifts the equilibrium of topo 2 activities to decatenetion of homological and heterological chromosomes. Spindle poisons like colcemide, vinblastine and nocodazole prevent anaphase onset: only at the beginning of anaphase one might observe perfect segregation of chromosomes; but short duration of this period (few minutes) leaves little chance to get a sufficient proportion of perfect karyotypes without clumps both in the case of blocked and unblocked cells.
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