Optimization of conditions for isolating chromosomes in flow sorting

Factors for purity and efficiency of flow chromosome sorting were analysed on the base of quantitative analysis. The sorting rate and relative purity of individual chromosome fractions are determined both by the quality of initial chromosome suspension, instrument parameters and gate position on experimental histograms. The described procedure of analysing sorting efficiency and fraction purity allowed to formulate general tips for optimization of sorting conditions depending on a given strategy: maximization of quantity of obtained material, or achieving maximum purity for sorted fraction. The analysis is carried out on the bases of chromosome distribution parameters: their relative halfwidths and distances. These parameters can be obtained by the quantitative analysis programs for flow cytometry data. It is shown that the critical parameter for sorting purity is the level of contaminated objects in a zone of sorted chromosome signal registration. In addition, the fraction purity depends on the cover extent between different chromosome distributions. Created procedure allows to build up nomograms linking the sorting efficiency with fraction purity, depending on the position of sorting gates. These monograms permit to determine the position of sorting gates in relation to one or another strategy: 1) maximum rate for chromosome material obtaining, 2) maximum fraction purity, or 3) compromise between the two. The presented analysis allows to optimize the chromosome sorting process for subsequent genome investigations providing chromosome material with controlled characteristics.