Chromosome Analysis and Sorting Using Conventional Flow Cytometers

The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. (c) 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Cell culture and metaphase harvest of suspension cell lineAlternate Protocol 1: Cell culture and metaphase harvest of adherent cell lineBasic Protocol 2: Preparation of chromosome suspension using polyamine isolation bufferBasic Protocol 3: Staining chromosomes with DAPI and propidium iodideAlternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3Basic Protocol 4: Bivariate flow karyotyping on a cell analyzerBasic Protocol 5: Bivariate flow karyotyping on a cell sorterBasic Protocol 6: Purification of flow-sorted chromosomes