FS
FlowSight
Abstract
The cell cycle is a series of orchestrated events
that leads to cell replication. It is characterized
by three interphase stages (Gap 1, S Phase,
and Gap 2) and a dividing stage called mitosis.
Mitosis is further divided into four primary
phases: prophase, metaphase, anaphase and
telophase. Progression of cells through the cell
cycle is highly regulated through feedback loops
that control cell growth via cyclin-dependent
kinases (CDKs). Activation of CDKs mark the
progression from one stage of the cell cycle to
the next and therefore offer opportunities to
intervene in cancer and other cell proliferative
disorders. A detailed assessment of the cell cycle
and mitosis are essential to the development of
CDK-targeted therapies. This application note
describes the use of the FlowSight for cell cycle
analysis via the classical assessment of DNA
content as well the calculation of the mitotic
index via the assessment of DNA condensation
in nuclear images.
Frequency
Cell Cycle
Measuring Cell Cycle and Mitotic Index with the FlowSight
Total Intensity DAPI
DNA content histogram of THP-1 cells fluorescently labeled with DAPI and
imaged on the FlowSight. The data were analyzed using Amnis' IDEAS
software and were exported in FCS 3.0 format to FlowJo to demonstrate
compatibility. The total intensity of each DAPI-stained nuclear image is
proportional to DNA content and was used to generate the histogram
showing G0/G1 (green), S (red) and G2/M (blue) stages of the cell cycle.
Example cell images of each stage are shown below.
Measuring Cell Cycle
Images From the Cell Cycle
Cells from the monocyte line THP-1 were fixed in
2% PFA, permeabilized with 0.1% Triton X-100
and fluorescently labeled with 4’,6-diamidino2-phenylindole (DAPI). DAPI stoichiometrically
binds to T-A rich regions of DNA and allows for
quantitative measurements of DNA content. Data
was acquired from 10,000 cells using a FlowSight
equipped with a 405nm laser for DAPI excitation.
The imagery of each cell included brightfield
(transmitted light), darkfield (side scatter), and
DAPI fluorescence. The total intensity of the DAPI
signal from each nuclear image was plotted in a
histogram, which shows a large G0/G1 peak of
cells with 2N DNA content, S phase cells that are
synthesizing DNA, and a smaller G2/M peak of
cells wtih 4N DNA content.
Gap 1
S Phase
Gap 2
Mitotic
Images of Mitotic Cells
Peak Pixel Intensity DAPI
Prophase
Metaphase
Anaphase
Total Intensity DAPI
Measuring Mitotic Index
Each phase of mitosis exhibits morphologically distinct
DNA staining patterns. In prophase, the nuclear envelope
is dissolved and DNA condenses into chromosomes.
Metaphase is characterized by chromosomal alignment to
the metaphase plate, followed by anaphase where sister
chromosomes are actively pulled towards the poles of the
cell. During telophase the cell membrane begins pinching
together to form two daughter cells. All phases of mitosis
are associated with condensed chromatin, which leads to
higher peak pixel intensities in the nuclear imagery. As a
result, the number of cells in mitosis (the mitotic index) can
be found simply by gating those cells in G2/M that exhibit
high peak pixel intensities, as shown above. With the THP-1
cell line, the "Mitotic" gate on the plot contains 4.6% of
the total. Visual inspection of the nuclear imagery of cells
in the Mitotic gate reveals cells in each of the four primary
phases of mitosis.
Telophase
Conclusion
A common application of conventional flow cytometry is cell
cycle analysis. Here, the FlowSight imaging flow cytometer
is used to perform a standard cell cycle analysis and to
further calculate the mitotic index using a simple image
feature available in real-time during data acquisition. The
high fluorescence sensitivity and linearly of the FlowSight
combined with its ability to generate imagery of every cell
results in superior quantitative flow cytometry and offers
visual verification of all results.
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