application note
Rapid analysis of cell cycle phases using
acumen® Cellista
key benefits
introduction
methods
acumen Cellista provides
the fastest way to perform
multicolour cell-based imaging
assays. It can:
The effect of compounds on the cell cycle is
widely studied in the drug discovery industry.
The cell cycle is divided into two main “phases”:
interphase and mitosis. Interphase is where the
cell prepares for mitosis and it is divided into
three sub-phases: G1, S, and G2. Movement
across these phases is regulated by a number
of complex signalling pathways. Mitosis is where
the chromosomes in the nucleus separate into
two identical sets (cell division).
cell treatment
n
screen up to 400,000 whole
wells a day
n
normalise to total cell number
n
multiplex cell cycle alongside
receptor or pathway signalling
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use a wide range of
commercially available dyes
with 405, 488, 561 or 640 nm
excitation
Traditionally, cell cycle assays have been carried
out using fluorescent dyes to stain for nuclear
content using flow cytometry methods to quantify
the amount of DNA. More recently however,
research groups are using fluorescence imaging
systems such as acumen Cellista for these
studies, allowing rapid high content screening of
the effect of compounds on the cell cycle.
In this application note we demonstrate
the use of fluorescent dyes to analyse the
cell cycle phases of adherent HeLa cells in
384‑well microplates using TTP Labtech’s
acumen Cellista. Of major significance is the
ability to analyse adherent cells in situ, which
provides valuable secondary information
by preserving morphological changes that
may have occurred during drug treatment.
In addition, multiplex assays (e.g. analysis of
the cell cycle alongside receptor or pathway
signalling) in microplates eliminate the
secondary effects associated with the harsh
washing and repeated cell re-suspension
required for analysis using flow cytometry.
The acumen Cellista laser scanning imager
offers rapid (under 5 minutes per plate), whole
well image acquisition in a wide range of plate
types (96- to 1536-well plates). acumen Cellista
enables a wide range of fluorescent reagents to
be combined in multicolour, multiplexed assays
as it is equipped with a choice of 405, 488, 561
and 640 nm lasers and able to simultaneously
acquire up to four channels of fluorescence data
per laser.
HeLa cells were plated out in 1536-well plates
and incubated with vinblastine overnight to arrest
cell division. Cells were stained with Hoechst to
determine DNA content.
image acquisition
Whole well TIFF images were acquired using
acumen Cellista’s 405 nm laser excitation for
Hoechst (Fig 1). acumen Cellista’s optics can
rapidly image the entire well area simultaneously
in multiple detection channels.
a
b
data analysis using histograms
Total DNA content was visualised and quantified
using histograms, plotting the total dye intensity
within each nuclei in cells across the entire well.
Fig 2 demonstrates a typical double peak, where
the lower intensity peak is identified as nuclei
containing a single copy of DNA and the higher
intensity peak is identified as cells containing two
copies of DNA. These peaks are classified as
G1 and G2/M respectively. A simple gating tool
on the histogram allows the user to move the
gates to the correct location on the histogram
to classify cells in G1, S or G2/M phases of the
cell cycle.
acumen Cellista application note / Rapid analysis of cell cycle phases / 2014
Fig 1. (a) Whole well image of untreated HeLa cells
stained with Hoechst. (b) Zoomed-in image showing
classified cells. G1 phase (yellow) and G2/M phase
(red).
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results
conclusion
further reading
As vinblastine arrests cells, it is important to
determine the percentage of cells in each cell
cycle phase, as absolute numbers may not give
meaningful results. Therefore, the total number of
cells is reported, along with the number of cells
in G1 and G2/M, allowing the percentage to be
calculated. To demonstrate acumen Cellista’s
multiplexing capabilities, the percentage of cells
in mitosis in the G2/M phase was identified using
a anti-phosphorylated histone H3 antibody,
followed by secondary labelling with a FITC
conjugate (Fig 3b).
This data demonstrates the ability of acumen
Cellista to rapidly analyse and assess cell cycle
phases. Since cell processing and labelling
is performed with microplates, this technique
is highly amenable to automation. The novel
design features of acumen Cellista used in these
studies permit whole well analysis at read times
compatible with primary screening campaigns
having daily throughputs of up to 400,000
compounds per day.
For further reading on cell cycle analysis
employing acumen Cellista see :
a
a
b
b
Fig 2. Cell cycle in HeLa cells. (a) Histogram of control cells with no treatment
showing a two peak distribution from a heterogeneous population of G1 phase
(yellow) and G2/M phase (red). (b) Histogram of vinblastine treated cells showing
a shift in the distribution of a population of cells, predominately in the G2/M phase
of cell cycle.
get in touch
Karlas, A. et al. (2010) Proc. Natl. Acad. Sci. U.S.
106: pp8671-6
Swanton, C. et al. (2009) P.N.A.S 106: pp8671-6
Kittler, R. et al. (2007) Nature Cell Biol. 9:
pp1401-12
Auld, D.S. et al. (2006) Methods in Enzymol. 414:
pp566-89.
Fig 3. a) Concentration-dependence of vinblastine treatment on the cell cycle.
(b) After incubation with vinblastine overnight, cells were fixed and stained
with Hoechst for cell cycle analysis and anti-phosphorylated histone H3 (FITC
secondary conjugate) to determine the mitotic cells in the G2 population.
TTP Labtech Ltd
TTP Labtech Inc
Melbourn Science Park, Melbourn
Hertfordshire SG8 6EE, United Kingdom
Tel: +44 (0) 1763 262 626
Fax: +44 (0) 1763 261 964
One Kendall Square, Suite B2303
Cambridge MA 02139-1594, United States
Tel: +1 (617) 494 9794
Fax: +1 (617) 494 9795
visit www.ttplabtech.com
or contact us at [email protected]
acumen Cellista application note / Rapid analysis of cell cycle phases / 2014
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