FS
FlowSight
Abstract
The Flow cytometric analysis of cell DNA
content is widely used for the estimation
of cell cycle phase distributions. Adding a
fluorescently-labeled cell cycle phase-specific
antibody to complement the DNA-binding dye
typically used for cell cycle analysis allows
for a robust bivariate cell cycle assay that can
accurately identify mitotic cells. By further
running such a bivariate analysis on an imaging
flow cytometer, the mitotic population can be
visually analyzed to identify and enumerate
cells in specific stages of mitosis. This study
demonstrates such an approach by pairing
the Amnis® FlowSight® imaging flow cytometer
with the EMD Millipore FlowCellect™ Bivariate
Cell Cycle Kit for G2/M Analysis.
Measuring Cell Cycle
The cell cycle can be divided into two distinct
stages. The first stage, Interphase, consists of
the G1, S, and G2 phases in which cells are active,
growing, and replicating DNA. Cell division takes
place in the second, M-phase or mitotic phase.
1.5
Normalized Frequency
Cell Cycle
Robust Cell Cycle Analysis Using the FlowSight Imaging Flow Cytometer
with the FlowCellectTM Bivariate Cell Cycle Kit for G2/M Analysis
1.2
0.9
0.6
G0/G1
S
0.3
0
G2/M
1e5
2e5
PI Intensity
3e5
Figure 1: Typical histogram of cell cycle analysis by DNA
content using a single fluorescent DNA-binding dye.
Researchers have employed various strategies to closely
interrogate each of the critical steps of the various phases
in the cell cycle. For example, analysis of cells in S-phase
provides a direct measurement of newly synthesized
DNA, while the ratio of cells in the G2 and M phases is
indicative of the number of cells undergoing mitosis. In
all, a comprehensive understanding of cell cycle behavior
provides useful information which can be important in
interpreting the intrinsic nature of cell proliferation and
assist in the development of anti-neoplastic agents.
Improving Cell Cycle Analysis
The FlowSight imaging flow cytometer operates like a
conventional flow cytometer but provides up to 12 images
of every cell, including brightfield, darkfield and up to nine
fluorescence channels. This unique combination provides
important new capabilities, such as visual verification
of each object acquired, and measurement of the spatial
location of fluorescence on, within, or between cells.
Visual verification removes the guesswork from gating and
can accelerate assay development and optimization by
providing visual confirmation of assay performance in any
individual cell or group of cells. Automated quantitative
measurement of fluorescence localization and morphology
enables a broad range of applications that would be
impossible by traditional flow cytometry with statistically
robust, quantitative image analyses that are not available
by conventional microscopy.
The FlowCellect Bivariate Cell Cycle Kit for G2/M Analysis
(cat. no. FCCH025103) simplifies the study of the cell
cycle by providing a cell cycle phase-specific antibody
(Anti-phospho-Histone H3 (Ser10) – Alexa Fluor 488
conjugated monoclonal antibody) in addition to a DNAbinding dye (Propidium Iodide) to yield a robust bivariate
analysis in flow cytometry applications. Moreover, the kit
includes a BrdU precursor to allow for BrdU measurement
by the conjugated antibody and an RNase reagent to
ensure that the PI intensity is directly proportional to DNA
content only. This method of analysis reveals not only the
distribution of cells in particular phases of the cycle, but
also provides insight into the molecular and functional
mechanisms associated within the cell cycle.
Example Bivariate Analysis
1.2
Single Cells
0.9
0.6
Doublets/Aggregates
Debris
0.3
0
0
300
600 900 1.2e3 1.5e3
Area Bright Field
Figure 2: Image analysis
parameters can be applied to
easily exclude debris and
aggregates from the analysis.
PH3 AF488 Intensity
Aspect Ratio Bright Field
The Amnis IDEAS software, provided with the FlowSight
cytometer, allows the user to click on any dot in a bivariate
scatter plot and visually verify whether or not it is an object
of interest. In figure 2, image parameters such as Area and
Aspect Ratio (the minor axis divided by the major axis, i.e.
shape of the object) are applied to the bright field imagery,
allowing debris and aggregates to be excluded from the
analysis with a high degree of confidence that the region for
Single Cells has been placed in exactly the right location.
1e5
FlowSight Mitotic Cell Images
Brightfield
Prophase
1528
Metaphase
43
Anaphase
488
Telophase
3144
pH3-AF488
PI
M Phase
1e4
1e3
0
0 1e5 2e5 3e5 4e5 5e5 6e5 7e5
PI Intensity
Figure 3: Discrimination
between G2 and M phase
cells by measuring the
phosphorylation of Histone
H3 on Ser10.
In figure 3, analysis for phosphorylation of Histone
H3 at Ser10 enables discrimination between cells in
G2and M phases of the cell cycle. Additionally, figure 4
demonstrates how the particular phases of mitosis can
be discerned by simply clicking on the events positive for
the phospho-Histone3 marker to visually inspect the Alexa
Fluor 488 image as well as the PI image. Hand-tagging
populations or performing further morphological
measurements on the nuclear imagery allows quantification
of each phase of mitosis if desired.
Figure 4: Example images of cells from each phase of the cell
cycle demonstrate the value of visual examination of images.
Both the morphology and the staining patterns clearly show the
transition through the stages of the cell cycle.
Conclusion
Coupling FlowSight data acquisition and analysis with
the EMD Millipore FlowCellect Bivariate Cell Cycle Kit for
G2/M Analysis provides a simple and robust method
for identifying, discriminating, and quantitating cells
between the G2 and M phases of the cell cycle, as well as
enumerating cells in various stages of mitosis. The ability
to visually and quantitatively distinguish cells undergoing
mitosis versus cells remaining in G2 phase of the cell cycle
provides a powerful tool to closely monitor cell cycle activity
at M-phase which impacts the study of diseases associated
with cell proliferation, apoptosis, and cancer.
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