Background: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb.
Methods: Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting.
Results: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot.
Conclusions: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.