GE Healthcare
Application Note 28-9327-11 AA IN Cell Analyzer 1000
Environmental control for automated live-cell
imaging with IN Cell Analyzer 1000
Key words: IN Cell Analyzer 1000 • Environmental
Control Module • cell tracking • Investigator •
Spotfire DecisionSite
The use of living cells for high-content and high-throughput
assays is becoming increasingly attractive for research and
drug discovery. When working with living cells, the ability to
control and maintain environmental conditions can be critical
for obtaining reliable and physiologically relevant assay results.
IN Cell Analyzer 1000 with the optional Environmental Control
(EC) Module provides an environmentally controlled sample
area, to help maintain cell health and viability during live-cell
imaging experiments on IN Cell Analyzer 1000. The EC Module
(in conjunction with the Temperature Control Module) provides
variable temperature control and CO2 supply in a humidified
environment designed to minimize evaporation.
Here, we describe the use of the EC Module to support cell
viability and proliferation for a range of cell types. Metrics
obtained from cells imaged in the chamber over three days
were compared with those achieved in a tissue culture incubator.
Cell behavior and phenotype were examined in both motility
and protein translocation assays. The results demonstrate
that the EC Module is enabling for live-cell imaging over extended
time periods, maintaining cell proliferation, viability, and
assay performance at the standards expected from a tissue
culture incubator.
Materials
Products used
Product­
Code Number
IN Cell Analyzer 1000 Kinetic instrument 28-4007-55
(includes Liquid Handling and Temperature Control Modules)
IN Cell Analyzer 1000 Environmental
28-4084-05
Control Module
IN Cell Investigator, Single Seat licence 28-4089-71
G1S-Cell Cycle Phase Marker (CCPM) Assay,
25-9003-97
Screening (cell line derived from U2-OS parental line)
Other materials required
CHO-M1 cell line
HUVEC cell line
(Clonetics) CC-2517
L929 cell line
(ATCC) CCL1
Ham’s F-12 medium (Gibco) 31765
EGM-2 medium (Clonetics)
Eagle’s Minimal Essential Medium (MEM) (Sigma) M2279
McCoy’s 5A médium (Sigma-Aldrich) M8403
Fetal bovine serum (FBS) (SAFC) 5N0581
Horse serum (Invitrogen) 26050-088
L-Glutamine (L-Gln) (Sigma-Aldrich) G7513
Penicillin-Streptomycin (Pen-Strep) (Sigma-Aldrich) P4333
EGM bullet pack (Clonetics) CC-4133
Geneticin (Sigma-Aldrich) G8168
Packard™ 96-well Viewplates™ (Perkin Elmer) 6005182
µClear™ 96-well plates (Greiner Bio-One) 655090
Hoechst™ 33342 (Molecular Probes) H3570
Propidium iodide (PI) (Molecular Probes) P3566
Roscovitine (Sigma-Aldrich) R-7772
Heraeus™ HERAcell™ 150 incubator (Heraeus) 51022391
Methods
Cell cycle
Cell culture
G1S-CCPM cells were seeded at 1.5 x 104 cell/ml onto replicate
96-well Viewplates, and incubated overnight in a tissue culture
incubator (37ºC, 5% CO2). At the start of the experiment,
roscovitine (30 µM final) was added to alternate wells in
a checkerboard pattern. At the same time, a low concentration
of Hoechst 33342 (0.5 µM final) was added to all wells to aid
cell identification during image analysis. Plates were then
transferred to either the EC Module or a tissue culture incubator
(both pre-equilibrated to 37ºC and 5% CO2), and incubated for
48 h. Images were acquired with a 10× objective (band-pass
480/40 ex. and 535/50 em. filters) to detect EGFP with a 1000
ms exposure time. For detection of Hoechst 33342, 360/40 ex.
and 535/50 em. filters were used with a 2000 ms exposure time.
Images were analyzed with IN Cell Analyzer 1000 Workstation
software using the Multi Target Analysis Module. The ratio of
nuclear and cytoplasmic intensities detected in the EGFP
channel was used to quantitate phase distribution of the
G1S-CCPM cells.
CHO-M1 and G1S-CCPM cell lines were grown in culture medium
(Ham’s F-12 or McCoy’s 5A, respectively) supplemented with
10% FBS, 2 mM L-Gln and 100 µg/ml of Pen-Strep. Geneticin
(500 µg/ml) was included in the McCoy’s medium to maintain
selective pressure on the G1S-CCPM cell line. HUVEC cells were
grown in EGM-2 medium supplemented with 2% FBS and EGM
bullet pack reagents. L929 cells were grown in MEM supplemented
with 10% horse serum, 20 mM L-Gln and 100 µg/ml of Pen-Strep.
Prior to image acquisition, all cells were maintained in a standard
humidified tissue culture incubator pre-equilibrated to 37ºC
and 5% CO2.
Cell viability and proliferation
CHO-M1 and HUVEC cells were seeded (CHO-M1 cells at 1 × 103
cells/well and HUVEC cells at 3 × 103 cells/well) in a checkerboard
pattern onto four replicate 96-well plates and maintained for
at least 24 h in a tissue culture incubator prior to the experiment.
For the duration of the 64 h time-course, replicate plates were
maintained: (a) in the tissue culture incubator; (b) at room
temperature (on the bench top); or (c) incubated in a preequilibrated EC Module. Following incubation, cells were
stained for 30 min with a mixture of 1 µM Hoechst 33342 (to
detect cell nuclei) and 8 µM PI (to detect cells with compromised
plasma membrane integrity). Following staining, cells were
imaged on an IN Cell Analyzer 1000 using a 10× objective
(360/40 ex. and 535/50 em. for Hoechst 33342; 620/60 ex.
and 535/50 em. for PI) with exposures of 500 ms for Hoechst
33342 and 400 ms for PI. The data were then analyzed for
total cell count (Hoechst-positive), live cell count (Hoechstpositive, PI-negative), and percent viability (percentage live
cells relative total).
Cell motility
L929 mouse fibroblasts were seeded at 5 × 104 cell/ml onto
96-well µClear plates (see “Cell culture”). To enable detection
of individual cells, Hoechst 33342 (1 µM final) was added prior
to image acquisition either in complete or serum-free culture
medium (to measure inhibition of movement in the absence of
serum). Following addition of dye, cells were transferred to the
pre-equilibrated EC Module (37°C, 5% CO2) and imaged every
15 min for 22 h (10× objective, 360/40 ex, 535/50 em, 2000 ms
exposure). Images were analyzed with IN Cell Investigator
software using the Cell Tracking analysis tool. The Cell Tracking
Spotfire™ guide was used to create plots of x-y position and
time-dependencies.
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28-9327-11 AA
Results
Cell viability and proliferation
Variations in the temperature and pH of culture media can have
profound effects on cell proliferation and health. Therefore,
count and viability of cells incubated in the EC Module for 64 h
(37ºC, 5% CO2) were compared with respective values obtained
from cells incubated in a commercial tissue culture incubator
over the same time period. Metrics were obtained from two
mammalian cell lines—one continuous cell line of rodent origin
(CHO-M1, derived from a Chinese hamster ovary parent cell
line) and one untransformed human cell line (human umbilical
vein epithelial cell, HUVEC).
After the 64 h incubation period, viability of the CHO-M1 cells
achieved with the EC Module was comparable to that achieved
using the tissue culture incubator (91.4% and 98.3% viable,
respectively). Viability of HUVEC cells was also comparable
between the two devices: 93.0% for the EC Module compared
with 92.6% for the tissue culture incubator (Table 1 and Fig 1).
Cell count values obtained using the EC Module were comparable
to or slightly higher than those obtained using the tissue culture
incubator (Table 1). As indicated in the table, when cells were
incubated in the absence of CO2, cell proliferation was minimal
after 64 h, demonstrating the value of performing extended
cell imaging experiments in an environmentally controlled
chamber. Further investigation into inter-incubator variability
(data not shown) demonstrated that cell viability and count
values obtained from replicate plates using the EC Module
consistently fall within 3 standard deviations of the mean
obtained using commercial tissue culture incubators.
Table 1. Comparative cell count and viability data*
CHO cell line
HUVEC cell line
Viable cell
count
Percentage
live cells
Viable cell
count
Percentage
live cells
T=0
402
99.2
197
98.4
Tissue culture
incubator
3468
98.3
667
92.6
IN Cell Analyzer
1000 EC Module
3437
91.4
887
93.0
Bench top
474
96.0
191
97.5
* Values obtained using the IN Cell Analyzer 1000 Environmental Control Module
and a commercial tissue culture incubator are presented. For reference, data
from cells incubated under ambient conditions on the bench top are also included.
the time course (Fig 2). Successful tracking required the use of
a nuclear dye to identify cells, combined with a cell tracking
tool designed to follow individual cells, including daughter cells
resulting from divisions (Fig 3). A relatively low concentration of
nuclear dye was used to avoid spurious induction of cell cycle
arrest at mitosis.
A bar chart displaying the average total migration distance
for the time course for the two treatment protocols (Fig 4)
demonstrates that conditions in the EC Module were sufficient
to support the cell motility assay. A statistically significant
difference was detected between cells incubated in the absence
or presence of serum, with a signal-to-noise ratio (S:N) of 3.36
achieved for the assay. (S:N was calculated as √|µp - µn/ (σp2 + σn2)| ,
where μ = mean signal, p = positive control, n = negative control
[serum-starved], and σ = standard deviation.)
(A) Cells stained with Hoechst 33342 (T = 0)
Fig 1. Plate maps representing CHO-M1 cell count data from Table 1. Panels
graphically represent the analysis data obtained from replicate 96-well plates
of CHO cells imaged (A) at time zero of the experiment; (B) after incubation for
64 h at room temperature (on a bench top); (C) after incubation for 64 h in the
IN Cell Analyzer 1000 EC Module; and (D) after incubation for 64 h in a standard
tissue culture incubator. Spot size represents cell count. Spot location
corresponds to the position of the well within a 96-well plate.
Cell motility assay
Cell migration requires the coordinated interaction of a number
of dynamic cell processes including signal transduction, rapid
cytoskeletal reorganization, integrin mobilization, and membrane
remodeling. Culture conditions, particularly temperature and
pH of the culture medium, have been shown to have a significant
effect on cell motility and phenotype1. We therefore tested
the ability of the EC Module to support a cell motility assay using
the mouse L929 fibroblast cell line.
Motility of L929 and other cell types is dependent on factors found
in serum. L929 cells were cultured in the presence or absence of
serum for 22 h in the EC Module, with images acquired every 15 min.
Application of cell tracking software allowed quantitation of cell
movement, velocity, and direction of individual cells throughout
(B) Scatter-plot displaying the x-y coordinates of individual cells.
Fig 2. Time-series data from a cell migration assay. Murine L929 fibroblasts were
maintained at 37ºC and 5% CO2 in the EC Module over a 22 h period. Images
were acquired every 15 min throughout the time course. (A) Cells stained with
Hoechst 33342 (1 µM) and imaged at time-zero of the incubation period; (B)
scatter-plot displaying the locations (x-y coordinates) of individual cells in the
same field-of-view over the 30 consecutive frames comprising the time series.
All points having the same cell identification number are connected by a line;
thus, each line of points represents the continuous time track for an individual
cell. The plot was created using Spotfire™ DecisionSite™ data visualization
software in conjunction with a Spotfire guide specifically designed for use with
the IN Cell Investigator cell tracking tool.
28-9327-11 AA
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(A)
Fig 4. Effect of serum on motility of murine L929 cells. L929 cells were incubated
in the EC Module in serum-free culture medium or in medium complete with
10% FBS. Error bars denote +/-1 SD from the mean of data acquired from 16
replicate wells. S:N = 3.36.
Cell cycle
Cell cycle progression is highly dependent on environmental
conditions because the culture medium determines cell growth
rate, which in turn governs the timing of cell cycle events. Here,
we describe the use of the EC Module to support a cell cycle
progression assay that relies on phase-dependent translocation
of a fluorescent reporter.
(B)
The G1S-CCPM reporter construct, expressed under the control
of the ubiquitin C promoter, comprises the enhanced green
fluorescent protein (EGFP) fused upstream of the helicase B
C-terminus (Fig 5a). The C-terminal domain contains cyclindependent kinase (CDK) phosphorylation sites and localization
motifs necessary for phase-dependent localization of the reporter
protein. The clonal cell line expressing the G1S-CCPM reporter
protein was derived from a human osteosarcoma parental line
(U2-OS). As the cell transitions from G1 through S phase and
into G2, the reporter protein translocates from the nucleus to
the cytoplasm (Fig 5b).
(A)
(C)
Fig 3. Analysis of individual cells using Investigator and Spotfire DecisionSite
software. The cell tracking tool in IN Cell Investigator was applied to monitor
individual cells, which were labeled with Hoechst 33342. (A) Time-lapse images
showing a selected region of the field-of-view where a particular cell (circled in
red), is dividing. Two daughter cells are observed at the 75 min time point; (B)
time tracks for the selected cell (labeled 17) and its two daughter cells (labeled
17.141 and 17.142) are presented in Spotfire using an x-y position scatter plot;
(C) a Spotfire plot displaying the time dependency of intensity measurements
derived from cell 17 and its daughters.
(B)
Fig 5. (A) Primary structure of the G1S-CCPM reporter construct. Expressed
under the control of the ubiquitin C promoter, the reporter consists of EGFP
fused upstream of the helicase B C-terminus, which governs phase-dependent
localization of the protein; (B) images showing localization of the reporter protein
during G1, S, G2, and mitosis.
28-9327-11 AA
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Replicate plates of G1S-CCPM cells in media containing no or
30 µM roscovitine were incubated, in either the EC Module or
a tissue culture incubator. Representative images acquired from
the two plates 48 h post-treatment show comparable cell
phenotypes and roscovitine-induced changes (Fig 6). Inspection
of the images suggests that roscovitine treatment results in
a change in localization of the reporter accompanied by a
decrease in cell number.
Analysis of the image data supports these observations.
Roscovitine, a CDK inhibitor, is known to arrest cells in G1
phase2. Consistent with this, the average cell count decreased
from 517 to 158 after 48 h of roscovitine treatment. Since G1
phase cells have relatively high nuclear-to-cytoplasmic ratios
compared to cells in other phases, they can be crudely classified
by thresholding on this parameter. Quantitation of the assay
using this convenient method of phase assignment showed
that the assay performed well in the EC Module, yielding a
S:N of 3.9 (based on 48 replicate samples per treatment condition).
In the absence of treatment, 11% of cells were detected in the
G1 phase category, with the percentage rising to 40% for cells
treated with roscovitine (Fig 7). Phase distribution results
obtained with the tissue culture incubator were comparable
– the proportion of cells in G1 phase increased from 9% to
36% with roscovitine treatment.
Untreated
Roscovitinetreated
Tissue culture incubator
IN Cell Environmental
Chamber
Fig 6. Representative images from a G1S-CCPM assay. Cells were incubated in
the absence or presence of 30 µM roscovitine for 48 h in either a tissue culture
incubator or the EC Module.
Conclusions
We have demonstrated the ability of the IN Cell Analyzer 1000
EC Module to maintain cell health and proliferation in four
different cell types—CHO-M1, HUVEC, L929, and U20S—for
incubation periods of up to 3 d. Data from a range of assays
confirm that cell appearance and behavior are indistinguishable
from what is achieved using a commercial tissue culture
incubator. The EC Module expands the capabilities of IN Cell
Analyzer 1000, enabling live kinetic assays to be conducted
over extended time courses.
References
1. Hartmann-Petersen R. et al. Individual cell motility studied by time-lapse video
recording: influence of experimental conditions. Cytometry 40 (4), 260–70 (2000).
2. Alessi, F. et al. The cyclin-dependent kinase inhibitors olomucin and roscovitine
arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity.
Experimental Cell Research 245 (1), 8–18 (1998).
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28-9327-11 AA
Fig 7. Comparative results from a G1S-CCPM Assay. Pie charts indicate the
cell cycle phase distribution of G1S-CCPM cells after incubation for 48 h in
the absence or presence of roscovitine in a tissue culture incubator or the EC
Module.
GE, imagination at work, and GE monogram are trademarks of General Electric Company.
The IN Cell Analyzer 1000 is the subject of US patent application number 10/514925,
together with other pending family members, in the name of GE Healthcare Niagara, Inc.
The IN Cell Analyzer 1000 and associated analysis modules are sold under use licenses
from Cellomics Inc. under US patent numbers US 5989835, 6416959, 6573039, 6620591,
6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993,
7060445, 7085765, 7117098; Canadian patent numbers CA 2282658, 2328194, 2362117,
2381334; Australian patent number AU 730100; European patent numbers EP 0983498,
1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568,
3576491, 3683591 and equivalent patents and patent applications in other countries.
All third party trademarks are the property of their respective owners. © 2008 General
Electric Company—All rights reserved. First published February 2008. All goods and services
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28-9327-11 AA 02/2008