Mammalian Chromosome Analysis and Sorting by Flow Cytometry

Abstract

AbstractThe analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single‐chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.This article was corrected on 18 July 2023. See the end of the full text for details.Basic Protocol 1: Mitotic block and cell harvestingBasic Protocol 2: Propidium iodide isolationSupport Protocol 1: Swelling testBasic Protocol 3: MgSO4 low‐molecular‐weight isolationBasic Protocol 4: Polyamine high‐molecular‐weight isolationSupport Protocol 2: Molecular‐weight determination of chromosomal DNABasic Protocol 5: Chromosome analysis and sorting