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Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Published on: December 31, 2015

Flow analysis and sorting of microchromosomes (<3 Mb).

Bee L Ng¹, Fengtang Yang, Nigel P Carter

¹The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom. bln@sanger.ac.uk

Cytometry. Part a : the Journal of the International Society for Analytical Cytology

|March 8, 2007

Summary

High-purity isolation of small human microchromosomes (2.7 Mb) is achievable using conventional flow cytometry. This method enhances detection sensitivity and sorting precision for microchromosome analysis.

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Area of Science:

Cytogenetics
Molecular Biology
Flow Cytometry

Background:

Analyzing and isolating microchromosomes (<3 Mb) requires sensitive detection and precise sorting.
Conventional flow cytometers' capability for high-purity microchromosome sorting was investigated.

Purpose of the Study:

To assess the feasibility of using a standard flow cytometer for high-purity isolation of 2.7 Mb microchromosomes.
To optimize detection and sorting parameters for small chromosomal elements.

Main Methods:

Microchromosomes were isolated from a human cell line using a modified polyamine buffer.
Chromosome preparations were stained with Hoechst and Chromomycin, then analyzed and sorted on a MoFlo sorter.
Purity was confirmed via reverse chromosome painting.

Main Results:

Gating on FSC vs. pulse width effectively discriminated clumps and debris.
This improved resolution of microchromosome peaks in bivariate Hoechst/Chromomycin fluorescence plots.
High-purity sorting of microchromosomes was achieved.

Conclusions:

Conventional flow sorters can effectively detect and sort microchromosomes (<3 Mb) with high resolution and purity.
The developed method enables precise isolation of small chromosomal structures.