Image vs. flow cytometry: DNA quantification in the cell cycle
Cell biology research at University of Copenhagen
The most common approach for determining the cell cycle stage is based on the quantification of cellular DNA content, which can be determined using fluorescent DNA-selective stains that exhibit emission signals proportional to DNA mass. DNA staining is typically performed on cells permeabilized with either non-ionic detergents or alcohol fixation. Traditionally, flow cytometry has been the standard method of choice for analyzing cell cycle distribution.
The cell cycle represents the most fundamental and important process in eukaryotic cells. Being an ordered set of events, culminating in cell growth and division into two daughter cells, the cell cycle is tightly regulated by defined temporal and spatial expression, localization and destruction of several cell cycle regulators. Cyclins and cyclin-dependent kinases (CDK) are major control switches for the cell cycle, causing the cell to move from G1 to S or from G2 to M phases.
In each population, cells are distributed among three major phases of cell cycle: G1/G0 phase (one set of paired chromosomes per cell), S phase (DNA synthesis with a variable amount of DNA), and G2/M phase (two sets of paired chromosomes per cell, prior to cell division).
There are a few ways to study cell cycle progression and regulation. Here, we present a comparison study between image and flow cytometry using cells permeabilized by either alcohol fixation or acid lysis.
Comparison of image & flow cytometric determination of DNA content
Exponentially growing Jurkat, CHO and U2OS cells were permeabilized by alcohol fixation, stained with DAPI and analyzed for DNA content by flow cytometry (upper row) and image cytometry (lower row). The acquired data were analyzed as described in the Methods section of the poster pdf. The green, yellow and cyan areas of the histograms represent G1, S and G2/M cells, respectively.
For all three cell lines, the DNA content histograms obtained with the image cytometer resemble those acquired with the flow cytometer. In all cases, the histograms display narrow G1 peaks with low coefficient of variance (CV).
See full dataset in the PDF poster
Dysregulation of the cell cycle is a distinct characteristic of cancerous cells and numerous chemotherapeutic drugs target cell cycle progression. To perform robust cell cycle analysis and to detect genomic abnormalities, such as aneuploidy, systems providing accurate and precise quantification of DNA content is required. Currently, flow cytometry is the gold standard for quantification of cellular DNA content.
In this study, we demonstrated that the NucleoCounter® NC-3000™ is highly accurate and precise for quantifying cellular DNA content when compared with the BD LSR II. Thus, we found a high degree of concordance between cell cycle distributions measured by the two cytometric systems.
Furthermore, we have compared two different methods for permeabilizing cells prior to DNA staining: Conventional alcohol fixation and a combination of mild acid and non-ionic detergent. The presented results show that the new acid lysis method allows for accurate quantification of DNA content. In general, cells lysed with acid provided lower coefficient variation (CV) of the G1 peak than cells fixed with alcohol.
In conclusion, cell cycle distributions quantified by image cytometry is accurate and precise when compared with flow cytometry, with the advantages of cost-effectiveness, robustness, and potential for morphological confirmation of the measured objects.
Olaf Nielsen, Anna Fossum and Soren Kjaerulff.