Abstract
The use of the DNA dyes Hoechst (HO) and chromomycin A3 (CA3) has become the preferred combination for the bivariate analysis of chromosomes from both human and animals. This analysis requires a flow cytometer equipped with lasers of specific wavelength and of higher power than is typical on a conventional bench top flow cytometer. In this study, we have investigated the resolution of chromosome peaks in a human cell line with normal flow karyotype using different combinations of DNA dyes on a number of flow cytometers available in a flow cytometry core facility. Chromosomes were prepared from the human cell line using a modified polyamine isolation buffer. The bivariate flow karyotypes of different DNA dyes combination; 4′‐6‐diamidino‐2‐phenylindole (DAPI) or Hoechst with propidium iodide (PI), obtained from different flow cytometers were compared to the reference flow karyotype of DAPI or Hoechst with chromomycin A3, generated from a Mo‐Flo cell sorter using laser power settings of 300 mW each of UV and 457 nm. Good chromosome separation was observed in most of the flow cytometers used in the study. This study demonstrates that chromosome analysis and sorting can also be performed on benchtop flow cytometers equipped with the standard solid state 488 and 355 nm lasers, using a DNA dye combination of DAPI or Hoechst with PI. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Highlights
THE analysis of chromosome by flow cytometry is termed flow karyotyping
The flow karyotype of the human lymphoblastoid cell line GM7016A labeled with DAPI and propidium iodide (PI) and measured using UV and 488 nm lasers is shown in Figures 1B(i), 2A(i), 3A(i), and 4A(i)
A new chromosome peak, not observed in the standard HO and chromomycin A3 (CA3) dye combination, was noted located near to the chromosome 8 peak (Fig. 1B(i), inset). This was flow sorted and verified to be normal chromosome 9 by chromosome painting. This chromosome 9* peak was observed with the DAPI and CA3 dye combination (Fig. 1A(i), inset) on the karyotype plot obtained from the Mo-Flo
Summary
THE analysis of chromosome by flow cytometry is termed flow karyotyping. To date, the most complete flow cytometric resolution of the human karyotype is achieved by staining with a combination of DNA dyes namely, Hoechst 33342 (HO) and chromomycin A3 (CA3) [1]. While there has been some success in the flow cytometric analysis of chromosomes stained with HO and CA3 using low power air-cooled lasers [5,6], the application of flow karyotyping remains limited to laboratories with flow cytometers that are equipped with lasers at these wavelengths. Lasers operating at such wavelength ranges are usually an expensive custom option. Acquired flow karyotypes using standard lasers and optical configuration with flow cytometers available in a flow cytometry core facility
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