Image cytometry vs. flow cytometry
Cell cycle analysis comparison:
NucleoCounter® NC-3000™ and BD LSR II
Introduction
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.
Method
Cell preparation
Cell lines: U2OS (human osteosarcoma cell line, ECACC #92022711), CHO (Chinese hamster ovary cell line, ECACC #85050302) and Jurkat (human leukemia T cell line, ATCC #CRL-2570) cells. Suspension cell line (Jurkat) was grown to a density of 5×10 5 cells/ml in RPMI + 6% FCS. The cell sample was divided into six T-flasks and half of the flasks were supplemented with 10 μM camptothecin (CPT). After 16 hours of incubation, the CPT-treated and untreated cells were harvested and analyzed for DNA content. Each of the samples were analyzed in duplicates. Adherent cell lines (U2OS and CHO) were grown to 90% confluency in RPMI + 6% FCS.
Cells were harvested by trypsination and the samples were divided into six T-flasks. The T-flasks were incubated for approximately 24 hours and at 75% confluence, half of the T-flasks (three for each cell line) were supplemented with 10 μM camptothecin (CPT). After a further 16 hours of incubation, CPT-treated and untreated cells were harvested and analyzed for DNA content. Each of the samples were analyzed in duplicates.
Cell staining
Cells were permeabilized and DAPI stained using two different methods. In method 1 (using the NC-3000™ Fixed Cell Cycle Assay), cells were permeabilized by alcohol fixation and after washing with PBS cells were stained with DAPI. In method 2 (using the NC-3000™ 2-Step Cell Cycle Assay), cells were permeabilized by acid lysis and DAPI stained without any prior washing steps.
Instrumentation
Parallel cell cycle analyses were performed on identical samples by traditional flow cytometry and image cytometry. Standard flow cytometry was performed using a BD LSR II cytometer configured with a 405 nm laser. 10.000 single cells were acquired collecting the DAPI blue fluorescence in the pacific blue channel. Image cytometry was performed using a NucleoCounter® NC-3000™. 10.000 single cells were acquired using either the instrument’s Fixed Cell Cycle Assay or its 2-Step Cell Cycle Assay.
Data analysis
To facilitate a direct comparison data obtained with the two different cytometers were exported in FCS format and analyzed using FlowJo software by Treestar. DNA content and cell cycle distribution for 10.000 single cells were automatically quantified and analyzed using the cell cycle-modeling algorithm Watson Pragmatic, version 7.6.5. Each data point in the columns shown below represents the average of six samples (three independent samples analyzed in duplicate).
Comparison of DNA histograms using the image cytometer and flow cytometer
Fig. 1. Representative examples of DNA histograms obtained from BD LSR II and NC-3000™
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. Green, yellow and cyan area of histograms represent, respectively, G1, S and G2/M cells.
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).
Image cytometer vs. flow cytometer using alcohol fixed cells
Fig. 2. Comparison of image cytometry cell cycle analysis and flow cytometric analysis using alcohol fixed cells. Concordance between NC-3000™ and BD LSR II on three different mammalian cell lines
Exponentially growing cells (untreated) and camptothecin treated cells (CPT treated) were permeabilized by alcohol fixation, stained with DAPI and analyzed for DNA content by flow cytometry (red-toned columns) and image cytometry (blue-toned columns). Acquired data were analyzed as described in the Methods section. Columns depict the percentage of the population determined to occupy each stage of the cell cycle. Sub-G1 represents cells with less than 2C DNA content. Each column represents the mean of six samples (three independent samples analyzed in duplicate). Standard deviation is indicated by an error bar.
For all three cell lines, there is a high degree of concordance between cell cycle distributions measured by flow cytometry and image cytometry. In general, the accuracy and precision of the NC-3000™ is comparable with BD LSR II. Thus, in the untreated Jurkat samples, the fraction of G1 cells is measured to 41.5±1.2% by BD LSRII and 40.3±1.2% by NC-3000™. Furthermore, in the CTP treated Jurkat samples the fraction of G1 cells is quantified to 10.8±1.4% by BD LSRII and 10.4±0.2% by NC-3000™. Good concordance is also observed for the other two investigated cell types.
The data demonstrate that NC-3000™ allows for accurate and quantitative investigations of subtle alterations in the cell cycle.
Image cytometer vs. flow cytometer using acid lysed cells
Fig. 3. Comparison of image cytometry cell cycle analysis and flow cytometric analysis using acid lysed
cells. Concordance between NC-3000™ and BD LSRII on Jurkat and U2OS cells
Exponentially growing cells (untreated) and camptothecin treated cells (CPT treated) were permeabilized by acid lysis, stained with DAPI and analyzed for DNA content by flow cytometry (red-toned columns) and image cytometry (blue-toned columns). Acquired data were analyzed as described in the Methods section. Columns depict the percentage of the population determined to occupy each stage of the cell cycle. Each column represents the mean of six samples (three independent samples analyzed in duplicate). Standard deviation is indicated by an error bar.
As observed with alcohol fixed cells, the two cytometric methods provided similar cell cycle distributions using acid lysed cells. For both Jurkat and U2OS cells there is a high degree of concordance between cell cycle distributions measured by flow cytometry and image cytometry. In general, the accuracy and precision of the NC-3000™ is comparable with BD LSRII. For example, for the untreated Jurkat samples the fraction of S-phase cells is measured to 44.9±0.6% by BD LSR II and 45.5±0.7% by NC-3000™. Furthermore, for the untreated U2OS samples the fraction of S-phase cells is quantified to 38.8±1.8% by BD LSR II and 40.5±1.1% by NC-3000™.
Image cytometer vs. flow cytometer mean of coefficient variation (CV) comparison
Fig. 4. Comparison of mean coefficient variation (CV) of the G1 peaks measured by flow and image cytometry
Exponentially growing cells were permeabilized by either alcohol (using the NC-3000™Fixed Cell Cycle Assay) or acid lysis (NC-3000™ 2-Step Cell Cycle Assay) and analyzed in parallel using flow cytometry (BD LSR II) and image cytometry (NC-3000™). Acquired data were analyzed as described in the Methods section. Columns depict the mean coefficient variation (CV) of the G1 peaks obtained from 6 samples (three independent samples analyzed in duplicate).
Note: no data for acid lysed CHO cells are available. The two cytometric systems provided relatively similar CVs of the G1 peaks. Compared to NC-3000™, BD LSRII produced somewhat lower CVs on cells fixed with alcohol. In contrast, NC-3000™ yielded lower CVs on cells lysed with acid than BD LSRII. In general, cells lysed with acid provided lower CVs than cells fixed with alcohol.
Conclusion
Dysregulation of the cell cycle is a distinct characteristic of cancerous cells and numerous chemotherapeutic drugs target cell cycle progression. In order 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 have compared an image cytometer, NucleoCounter® NC-3000™, and a flow cytometer, BD LSR II, with respect to quantifying DNA content, thus determining cell cycle distributions. The data demonstrate that the 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. In method 1, cells are permeabilized by conventional alcohol fixation. In method 2, a new rapid approach is employed where cells are lysed with 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.
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Authors:
Olaf Nielsen, Professor (Department of Biology, University of Copenhagen)
Anna Fossum, Staff scientist (BRIC, FACS core, University of Copenhagen)
Søren Kjaerulff, PhD (ChemoMetec A/S)
Article based on original comparison by the above authors and edited for the web.
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