Image vs. Flow cytometry – A comparison of apoptotic events
Cell biology research at university of copenhagen
Cell death by apoptosis is a complex and tightly regulated process in which a cell orchestrates its own destruction in response to specific internal or external stimuli. Dysregulation of apoptosis can lead to various physical disorders such as cancer and autoimmunity. Information about apoptosis is commonly obtained either by flow cytometry or fluorescence microscopy.
Flow cytometry provides quantitative information for thousands of cells but does not allow for visualization of the cells. In contrast, fluorescence microscopy provides visual information, but does not allow for easy quantitative measurements of large cell populations. Image-based cytometry bridges the gap between these two technologies and allows for simultaneous quantitative analysis and visualization of thousands of cells.
Comparison of image & flow cytometric determination
Cells respond to apoptotic signals by initiating intracellular processes that result in characteristic physiological changes. Among these changes are externalization of phosphatidylserine to the cell surface, depolarization of the mitochondrial membrane, activation of proteases, compaction and fragmentation of nuclear chromatin, loss of cell membrane integrity, and cell shrinkage.
We have examined three different markers commonly used for detection of apoptotic cells:
- Phosphatidylserine translocation – using FITC conjugated Annexin V peptide
- Depolarization of the mitochondrial membrane – using JC-1 dye
- Activation of Caspase 3/7 – using FAM-conjugated DEVDFMK peptide
Exponentially growing cells (untreated) and camptothecin (CPT)-treated cells were labeled with FITC-Annexin V (a peptide binding externalized phosphatidylserine), Hoechst-33342 and propidium iodide (PI).
- A) The fraction of Annexin V positive and PI positive cells was determined by flow cytometry (upper panel) and image cytometry (lower panel). Each pie represents the mean of six samples (three independent samples analyzed in duplicate).
- B) Micrograph of CPT treated CHO cells. Micrograph was produced by superimposition of images captured in, respectively, the blue (Hoeschst-33342), green (FITC-Annexin V) and red (PI) channels of the NucleoCounter® NC-3000™. Arrows indicate the four different populations present in the samples. The optical magnification of the instrument is 2×.
Conclusion
“We have employed an image-based cytometer, the NucleoCounter® NC-3000™, for quantifying different events in the apoptotic process. Compared to flow cytometric analyses (BD LSRII), NC-3000™ demonstrated accurate and precise determination of phosphatidylserine translocation, Caspase 3/7 activation and depolarization of the mitochondrial membrane.”
Olaf Nielsen, Professor at the Copenhagen of University.
Currently, flow cytometry stands as the gold standard for gathering quantitative information about large cell populations. In this study, we have compared an image cytometer, NucleoCounter® NC-3000™, and a flow cytometer, BD LSRII, with respect to quantifying different events in the apoptotic process.
In the comparison study we have measured phosphatidylserine externalization, collapse of the mitochondrial membrane potential and activation of Caspase 3/7, all of which are well-recognized markers for apoptotic cells.
For all three markers, the NC-3000™ was accurate and precise for quantification of apoptotic cells when compared with BD LSRII. Thus, we found a high degree of concordance between the fractions of apoptotic cells measured by the two cytometric systems.
In summary, the NC-3000™ image cytometer presents a new technology that allows for accurate and quantitative investigations of subtle alterations in cell homeostasis in large populations. In contrast to conventional flow cytometry, NC-3000™ enables the user to validate the sample by visual inspection of the individual cells.
Authors
Olaf Nielsen, Anna Fossum and Soren Kjaerulff.