BIOIMAGING
APPLICATION NOTE
BD Biosciences
Fluorescent Protein Organelle Biomarkers are Beneficial in LiveCell and Fixed-Cell–Based Imaging Applications
INTRODUCTION
We have expanded the BD™ Bioimaging Certified reagents
portfolio to include eight BD Pharmingen™ FP organelle
vectors. Fluorescent protein (FP) tags, which are expressed
as fusion proteins to a gene or domain of interest, have
been used in numerous cell applications such as monitoring
protein trafficking, detecting gene activation, tracking
cellular differentiation, and as subcellular organelle and
cytoskeletal component biomarkers. These markers can be
used alone or multiplexed with other probes for high-content
applications.
High-content imaging is a technology that can provide
considerable insight into the behavior of cells; and therefore,
is becoming an increasingly common tool in drug screening,
translational biology, and other biological research
laboratories. Both live-cell and fixed-cell (endpoint) imaging
are used in high-content analysis. Live-cell assays can take
advantage of time-lapse fluorescent microscopy to provide
detailed phenotypic and quantitative information. However,
live-cell assays can be technically challenging to develop and
implement. Endpoint assays, which provide a single snapshot, are typically more user-friendly because they offer
convenient stopping points, and the assays can be batch
processed or automated to optimize throughput.
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Assays for high-content imaging or other quantitative
imaging applications typically go through a development
phase prior to implementation. During assay development, it
is often desirable to perform assays in a live-cell environment
to optimize time points for the experimental outputs,
and then transition the assay into screening mode as an
endpoint assay. In this scenario, it is preferable to use the
same fluorescent probes during assay development (live cell)
and screening (endpoint). However, due to the physical,
chemical, and biochemical properties of some fluorescent
probes this is not always possible. FPs, however, can be used
in both live-cell and endpoint applications.
We tested the eight BD Pharmingen FP organelle vectors in
both live and fixed mammalian cell imaging applications
and verified their functional location using colocalization
studies. In addition, a stable cell line expressing the Red
FP Mitochondrion vector was used to monitor apoptosis
in a live-cell kinetic experiment. This cell line was also
multiplexed with a dye and an antibody in a high-content
endpoint apoptosis assay.
Bioimaging Application Note – FP Organelle Vectors
Introduction
(continued)
The availability of such versatile tools extends the possibilities
for developing fluorescent-based assays that monitor not only
protein location and trafficking within cellular structures but
also the dynamics of cellular organelles. The Red or Green FP
organelle markers described here can be transiently or stably
transfected alone and in combination to obtain single-color or
multicolor assays. Both fluorescent proteins are easily detected
in living and fixed cells using a BD Pathway™ high-content cell
analyzer or other fluorescent cell analysis platform.
METHODS
Cell culture
Human cervical cancer (HeLa) cells (ATCC, CCL-2) were
grown in 37°C, 5% CO2 humidified incubators and maintained
in Minimum Essential Medium Eagle supplemented with 10%
fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium
pyruvate, 100 Iu/mL penicillin, 100 µg/mL streptomycin,
(Mediatech, 10-010-CV, 35-010-CV, 25-005-CI, 25-000-CI, and
30-002-CI, respectively).
Transfection and isolation of stably transfected cell
populations
HeLa cells (3x105 cells in 2 mL of medium) were plated into
wells of a 6 well plate (Cat. No. 353046). After 24 h, they were
transfected with 1 μg of FP organelle plasmid DNA using 3 µL
of FuGENE® 6 Transfection Reagent (Roche Applied Science,
11814443001) according to manufacturer’s recommendations.
After 48 h, transfected cells were selected using 0.5 mg/mL
G418 sulfate (Mediatech, 30-234-CR) and kept in selection
medium for two weeks. Then, a population of the 10% most
highly expressing cells (based on fluorescence intensity) was
isolated by cell sorting using a BD FACSAria™ cell sorter (Cat.
No. 333888).
Transient cotransfection on glass cover slips
HeLa cells (3x105 cells in 2 mL of medium) were plated on
glass cover slips (18 mm square) in wells of a 6 well plate.
After 24 h, cells were transfected with 0.5 μg of each of two FP
organelle plasmid DNA using 3 µL of FuGENE 6 Transfection
Reagent. After 48 h, cells were fixed with pre-warmed (37°C)
3.7% formaldehyde (Sigma-Aldrich, 252549) for 10 min,
washed three times with phosphate buffered saline (PBS),
and mounted on slides using Vectashield mounting medium
containing DAPI (Vector Laboratories, H-1200).
Transient transfection in 96 well bioimaging plates
HeLa cells (1x104 cells/well in 100 µL of medium) were seeded
in 96 well bioimaging plates (Cat. No. 353219). After 24 h,
the medium was removed and replaced with a transfection
mixture that contained 50 ng of FP organelle plasmid DNA and
0.15 µL of FuGENE 6 Transfection Reagent in a total of 100
µL medium/well. After 48 h, cells were imaged or processed
for verification of subcellular localization of the FP organelle
marker.
Verification of subcellular localization
Volumes for all procedures except for antibody labeling were
100 µL/well. Antibody labeling was performed using 50 µL/
well. Antibodies were diluted in blocking buffer. Hoechst 33342
(Invitrogen, H3570) was used at 2 µg/mL.
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Nuclei – Culture medium from live-cell cultures was replaced
with Hanks Balanced Salt Solution (HBSS) containing
Hoechst, and the cells were imaged.
Mitochondrion – Culture medium was replaced with fresh
medium containing 200 nM of MitoTracker® Deep Red
633 (Invitrogen, M22426). Cells were incubated for 45 min,
washed twice with HBSS, then HBSS containing Hoechst was
added, and the cells were imaged.
Endoplasmic Reticulum (ER) – Cells were fixed as above, and
permeabilized using 0.1% Triton™ X-100 (Sigma, T-9284)
diluted in PBS, for 5 min, washed three times, blocked for 30
min in 3% FBS in PBS, and washed once. Cells were labeled
with 0.5 µg/well anti-ERp61 antibody (Cat. No. 612585) for
1 h at room temperature and washed three times. Cells were
incubated in 5 µg/mL of Alexa Fluor® 647 labeled goat antimouse IgG (Invitrogen, A21236) for 1 h in the dark. Cells
were washed three times, PBS containing Hoechst was added,
and the cells were imaged.
Actin – Cells were fixed as above, and permeabilized for
5 min in 90% methanol (-20°C). Cells were washed and
blocked as above. Cells were labeled with a 1:10 dilution
of BD™ Bioimaging Certified Alexa Fluor® 647 Mouse
anti-Actin (Cat. No. 558624) for 1 h in the dark. Cells were
washed three times, and PBS containing Hoechst was added,
and the cells were imaged.
Golgi Apparatus – Cells were fixed, Triton permeabilized, and
blocked as above. Cells were labeled with a 1:10 dilution of
Bioimaging Certified Alexa Fluor® 647 Mouse anti-GM130
(Cat. No. 558712) for 1 h in the dark. Cells were washed
twice, PBS containing Hoechst was added, and the cells were
imaged.
Peroxisomes – Cells were fixed, Triton permeabilized, and
blocked as above. Cells were labeled with 0.5 µg/well antiperoxisomal membrane protein 70 antibody (Invitrogen,
S34201) for 1 h at room temperature and washed three times.
Cells were incubated in 5 µg/mL of Alexa Fluor® 647 labeled
goat anti-rabbit IgG (Invitrogen, A21245) for 1 h in the dark.
Cells were washed three times, PBS containing Hoechst was
added, and the cells were imaged.
Apoptosis assays
Live-cell assay – HeLa cells stably transfected with the Red
FP-Mitochondrion vector were seeded (1x104 cells/well) into
96 well imaging plates. Sixteen to 24 h later, plates were
placed into the pre-equilibrated environmental chamber of a
BD Pathway 855 cell analyzer. Images were obtained prior to
addition of 1 µM (final concentration) staurosporine to the
wells by the BD Pathway 855 on-board fluidics, and every 45
min thereafter for 225 min.
Fixed-cell assay – HeLa cells stably transfected with the Red
FP-Mitochondrion vector were seeded (1x104 cells/well) into
96 well imaging plates. Sixteen to 24 h later, staurosporine
(1 µM final concentration), or vehicle control was added to
duplicate wells at hourly increments over 4 h. After treatment,
cells were fixed and washed, as above, and permeabilized
with BD Perm/Wash™ buffer (Cat. No. 554723) for 30
min at room temperature. The cells were incubated with
a 1:10 dilution of Bioimaging Certified Alexa Fluor® 647
Mouse anti-cleaved PARP antibody (Cat. No. 558710) in BD
Perm/Wash buffer at 4°C overnight. Cells were washed three
times, PBS containing Hoechst was added, and the cells were
imaged.
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Bioimaging Application Note – FP Organelle Vectors
Imaging and analysis
Cells were imaged on a BD Pathway™ 855 cell analyzer (Cat.
No. 341036) using the objectives stated in the figure legends
and filter sets appropriate for the excitation and emission
spectra of the samples. Image and data analysis were performed
using BD Pathway software analysis tools. Cell populations
were analyzed for nuclear and cytoplasmic fluorescence
intensity and size.
The Green FP has an excitation maxima of 475 nm and an
emission maxima of 505 nm; the Red FP has an excitation
maxima of 558 nm and an emission maxima of 585 nm (Table
2).16, 17 The excitation and emission wavelengths of these FPs
make them suitable for detection using a wide range of cell
analysis instruments including the BD Pathway and other highcontent imaging platforms, standard fluorescent microscopes,
flow cytometers, cell sorters, and fluorescent plate readers.
Localization of FP vectors
RESULTS AND DISCUSSION
Organelle vectors
We describe a panel of eight vectors expressing fusion proteins
composed of Green or Red FPs and proteins that target various
cellular structures or organelles (Table 1). The FP fusions were
expressed under the control of the immediate early promoter
of cytomegalovirus, an efficient promoter in many cells.12 To
increase the translation efficiency in mammalian cells, a Kozak
consensus translation initiation site13 was introduced to the 5´end of the FPs open reading frame, and the FP sequence was
optimized with human codons.14 The FP fusion sequence was
followed by downstream SV40 polyadenylation signals.15
To confirm the FPs were targeted to the correct subcellular
location or organelle, HeLa cells were transiently transfected
with the various FP vectors and then co-labeled with a dye
or antibody known to target the same subcellular location
or organelle (Table 3). Location and organelle specific
confirmatory probes were selected such that the excitation
and emission spectra were non-overlapping with those
of the FP vectors. Images in the respective channels were
captured and color channel merged (pseudocolored) to
verify the colocalization of the FP probe with the confirming
probe (Figure 1). In all cases except the nuclear markers,
colocalization of staining was represented by a yellow to
orange color (depending on the level of expression of the
Table 1. Bioimaging Certified FP organelle marker vectors.
Vector Name and Location
Fusion Tag
Ref.
Cat.
No.
Green FP – Actin
C-terminal whole actin fusion
1,2
558721
Green FP – Golgi
N-terminal human ß 1,4-galactosyltransferase (aa 1-81)
3,4
558719
Green FP – Mitochondrion
N-terminal mitochondrial targeting sequence of cytochrome c oxidase
subunit VIII
5
558718
Green FP – Nucleus
C-terminal SV40 Large T antigen nuclear localization signal (in
triplicate)
6,7
558720
Red FP – Endoplasmic
Reticulum
N-terminal calreticulin ER targeting sequence + C-terminal KDEL ER
retention signal
8,9
558725
Red FP – Mitochondrion
N terminal mitochondrial targeting sequence of cytochrome c oxidase
subunit VIII
5
558722
Red FP – Nucleus
C-terminal SV40 Large T antigen nuclear localization signal (in
triplicate)
6,7
558723
Red FP – Peroxisome
C-terminal peroxisomal targeting signal 1 tripeptide (SKL)
10, 11
558724
Table 2. Green and Red FP excitation and emission maxima.
Fluorescent Protein
Excitation Maxima
Emission Maxima
Green
475 nm
505 nm
Red
558 nm
585 nm
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Bioimaging Application Note – FP Organelle Vectors
Table 3. Colocalization of confirmatory probes.
FP Vector
Confirmatory Probe
Mechanism of Confirmatory
Probe
Ref.
Figure 1 Panel and Color
of Colocalized Probes
Red - Nucleus
Hoechst 33432 dye
DNA binding dye
18
Panel A, Magenta
Green - Nucleus
Hoechst 33432 dye
DNA binding dye
18
Panel B, Cyan
Red - Mitochondrion
MitoTracker dye
Accumulates in mitochondria
19
Panel C, Yellow - orange
Green - Mitochondrion
MitoTracker dye
Accumulates in mitochondria
19
Panel D, Yellow - orange
Red - Endoplasmic
Reticulum
Anti-ERp61 antibody
Recognizes an ER chaperone
protein
20
Panel E, Yellow - orange
Green - Actin
Anti-actin antibody
Recognizes actin filaments
21
Panel F, Yellow - orange
Red - Peroxisome
Anti-PMP70 antibody
Recognizes a peroxisomal
membrane protein
22
Panel G, Yellow - orange
Green - Golgi
Anti-GM130 antibody
Recognizes a 130-kDa golgi matrix
protein
23
Panel H, Yellow - orange
Figure 1. Verification of FP organelle marker location in
transiently transfected cells. Cell images were color channel
merged. The red FPs were pseudocolored red, the confirmatory
stain was pseudocolored green (Panels C, E, and G), or blue
(Panel A). The green FPs were pseudocolored green, the
confirmatory stain was pseudocolored red (Panels D, F, and H),
or blue (Panel B). Hoechst dye was pseudocolored blue. Panel
A, Red FP-Nucleus and Hoechst. Panel B, Green FP-Nucleus
and Hoechst. Panel C, Red FP-Mitochondrion and MitoTracker
Deep Red. Panel D, Green FP-Mitochondrion and MitoTracker
Deep Red. Panel E, Red FP-Endoplasmic Reticulum and antiERp61. Panel F, Green FP-Actin and anti-actin. Panel G, Red
FP-Peroxisome and anti-PMP70. Panel H, Green FP-Golgi and
anti-GM130. Cells were imaged using a 40x objective (0.9 NA)
in confocal mode.
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Bioimaging Application Note – FP Organelle Vectors
FP in each cell) in the merged images. For the nuclear
markers, colocalization was represented as magenta for the
Red FP and cyan for the Green FP. In addition to general
nuclear localization, the FP nuclear markers showed distinct
localization to nucleoli. Cells that were not transiently
transfected with the FP vectors showed staining only with the
non-FP confirmatory probe.
Live-Cell and Fixed-Cell Imaging
To show the utility of the FP organelle vectors in live-cell
imaging, cells were transiently transfected with each marker
and were imaged in the environmental chamber of the BD
Pathway™ 855 cell analyzer. Figure 2 shows live-cell confocal
images of HeLa cells expressing the four Green FP organelle
markers. Cells stably expressing the Green FP-Actin exhibited
specific signal in actin filaments (Figure 2, Panel A). Similarly,
cells stably expressing the Green FP-Nucleus were imaged
and the signal localized only in the nucleus (Figure 2, Panel
B). Cells expressing the Green FP-Golgi fusion had signal
localized to small cytoplasmic golgi structures around the
nucleus (Figure 2, Panel C), and cells expressing the Green
Figure 2. Live-cell imaging of transiently transfected Green FP
tagged organelle markers. Live-cell images were color channel
merged. The green FP signal was pseudocolored green and Hoechst
staining was pseudocolored blue. Panel A, Green FP-Actin; Panel
B, Green FP-Nucleus; Panel C, Green FP-Golgi; Panel D, Green FPMitochondrion. Cells were imaged using a 40x objective (0.9 NA) in
confocal mode.
FP-Mitochondrion had signal in the mitochondria (Figure 2,
Panel D). Non-transfected cells showed only Hoechst staining
and no FP-specific signal.
Red FP vectors are also suitable for live-cell imaging, as shown
in Figure 3. HeLa cells stably expressing the Red FP-Nucleus
exhibited fluorescence in the nucleus (Figure 3, Panel A). Cells
expressing the Red FP-Mitochondrion fusion had signal in the
mitochondria (Figure 3, Panel B). Stably transfected cells with
the Red FP-Peroxisome construct had a punctate fluorescent
pattern corresponding to the peroxisome organelle distribution
throughout the cytoplasm of the cells (Figure 3, Panel C).
Finally, stable expression of the Red FP-Endoplasmic
Reticulum fusion in HeLa cells allowed visualization of the ER
network located in the cytoplasm of the cells (Figure 3, Panel
D). Again, non-transfected cells exhibited Hoechst staining but
not FP-specific signal.
Cotransfection of more than one FP vector is also possible.
This may be especially useful for studying protein trafficking
between subcellular compartments. HeLa cells were transiently
transfected with 2 FP vectors and imaged. Representative
Figure 3. Live-cell imaging of transiently transfected Red FP
tagged organelle markers. Live-cell images were color channel
merged. The red FP signal was pseudocolored red and Hoechst
staining was pseudocolored blue. Panel A, Red FP-Nucleus; Panel B,
Red FP-Mitochondrion; Panel C, Red FP-Peroxisome; Panel D, Red FPEndoplasmic Reticulum. Cells were imaged using a 40x objective (0.9
NA) in confocal mode.
Figure 4. Imaging of dual transiently transfected FP tagged organelle markers in fixed cells. Fixed-cell images were color channel merged.
The green FP signal was pseudocolored green, the red FP signal was pseudocolored red, and DAPI staining was pseudocolored blue. Panel A, Green
FP-Golgi and Red FP-Nucleus; Panel B, Green FP-Nucleus and Red FP-Mitochondrion; Panel C, green FP-Nucleus and Red FP-Peroxisome. Cells were
imaged using a 40x objective (0.9 NA) in confocal mode.
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Bioimaging Application Note – FP Organelle Vectors
images from cotransfection experiments are shown in Figure
4, Panel A shows the Green FP-Golgi and Red FP-Nucleus
markers where the green signal was localized in cytoplasmic
golgi apparatus and the red signal was localized in the nucleus.
The nucleus and mitochondria of cells expressing the Green
FP-Nucleus and the Red FP-Mitochondrion were labeled by
the FPs as shown in Panel B. Panel C shows cells cotransfected
with Green FP-Nucleus and Red FP-Peroxisome vectors.
Untransfected cells showed only DAPI staining.
Performing FP-based assays using fixed cells provides the
opportunity to multiplex the FP signal with antibody labeling.
This can provide multiple (confirmatory) readouts on a cell
process being investigated or enable simultaneous collection
of information on multiple cellular processes. When using
FP vectors in fixed-cell applications, we noted some loss in
fluorescent intensity post fixation. However, camera exposure
times for fixed cells transfected with FP vectors were in the
same range as other types of probes (1 to 500 ms exposures for
non-confocal images). Figure 5 shows a representative example
of the same field of view before and after formaldehyde
fixation. Exposure time for the live-cell image was 1 ms and
5 ms for the fixed image. There was no change in cellular
localization for any of the FP probes after fixation (or
permeabilization, data not shown). Some modest organelle
structural changes were noted (Figure 5, Panel B). These were
likely the result of the fixation process on delicate cell structures
Figure 5. Fixation of an FP organelle probe. HeLa cells stably
expressing the Red FP-Mitochondrion were imaged prior to formaldehyde
fixation (Panel A), and then the same field of view was imaged post
fixation (Panel B). Images were acquired with exposure times of 1 ms
(Panel A) and 5 ms (Panel B) with a 20x objective (0.75 NA) .
such as mitochondria. Overall, the data shows that cells
expressing FP vectors can be successfully imaged alive or after
treatment with fixative.
Assay development using FP vectors
We investigated the utility of HeLa cells stably expressing the
Red FP-Mitochondrion vector for detecting staurosporineinduced apoptosis in live and fixed cells. Staurosporine is a well
know inducer of apoptosis in HeLa cells and has been reported
to induce changes in the mitochondrial network morphology.24
For the live-cell assay, baseline images were obtained prior to
adding staurosporine, and then wells were imaged every 45 min
for 225 min after compound addition. Visual inspection of the
kinetic series showed dramatic cellular morphological changes
over time and distinct condensation of the mitochondrial
structures and the cytoplasm of the cells (Figure 6, Left).
Quantitative analysis of the average total mitochondrial area
(μm2) in the kinetic images (Figure 6, Right) was performed
by segmenting individual cells based on the fluorescence in the
mitochondria, and the analysis showed the time-dependent loss
of cytoplasmic area.
Next, we explored the use of HeLa cells stably transfected with
the Red FP-Mitochondrion vector in a multiplexed endpoint
assay. Once again, 1 µM staurosporine was used to induce
apoptosis over time. In this assay, in addition to the quantitative
data obtained from the FP mitochondrion tag, measurements
of a second apoptosis marker, cleaved poly(ADP-ribose)
polymerase (PARP), was multiplexed along with nuclear size
measurements based on Hoechst staining. The PARP protein
is involved in DNA repair and is cleaved by proteins that
are upstream in the apoptosis cascade, including caspase-3.25
Cleaved PARP was detected using a BD™ Bioimaging Certified
directly conjugated anti-cleaved PARP antibody. Figure 7,
Panels A – I show representative well images from 0, 2, and 4
h time points in the three channels (blue, green, red) imaged to
detect Hoechst staining of the nuclei, cleaved PARP labeling,
and Red FP-Mitochondrion labeling, respectively.
The red channel images show the same distinct cytoplasmic
morphology condensation over time (Panels D – F) as was seen
in the live-cell assay. Quantitation of the average cytoplasmic
size (in pixels) is shown in Panel J (for details, see Figure
8). As with the live-cell data, the most dramatic change in
Figure 6. Live-cell kinetics of apoptosis. Left, representative cropped images of the same field of view of HeLa cell expressing the Red FPMitochondrion marker taken prior to addition of 1 µM (final concentration) staurosporine (time 0) and for five subsequent 45 min time points. Cells
were imaged using a 20x objective (0.75 NA). Right, analysis of the average total mitochondrial area ± SEM, N=5.
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Bioimaging Application Note – FP Organelle Vectors
Figure 7. A multiplexed apoptosis
endpoint assay. Panels A – I, HeLa
cells stably expressing the Red FPMitochondrion marker were stained
with Hoechst and for cleaved PARP.
Representative images from wells
treated with vehicle control or 1 μM
staurosporine for 2 or 4 hours are
shown. Cleaved PARP staining is
pseudocolored green (Panels A – C),
the Red FP-Mitochondrion signal is
pseudocolored red (Panels D – E), and
Hoechst staining is pseudocolored blue
(Panels G – I). Montaged (2x2) images
were acquired using a 20x objective
(0.75 NA). Panels J – M show the
quantitative analysis of the time course
data. Bar graphs show the mean ± SD,
N=2.
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Bioimaging Application Note – FP Organelle Vectors
Figure 8. Cell segmentation parameters in a multiplexed endpoint assay. HeLa cells stably expressing the Red FP-Mitochondrion were stained
with Hoechst and for cleaved PARP after treatment with vehicle control (Panels A – C) or 1 µM staurosporine for 4 h (Panels D – F). One cropped field
of view (20x) is represented in Panels A – C and another in Panels D – F. Segmentation ROIs are shown as randomly colored cytoplasmic or nuclear
boundaries with their associated ROI number. In Panels A and D, cytoplasmic size was measured as a 50-pixel ring dilated out from the nuclear mask
(Panels B and E) but within the outer boundary of the cell’s cytoplasmic boundary, which was defined by the FP signal (dual channel two outputs
segmentation). Cells not expressing the FP were excluded from all calculations based on a user-defined minimum intensity threshold and do not display
ROIs. In Panel B and E, nuclear size was calculated using a nuclear mask identified by the Hoechst stain. Cleaved PARP signal was measured in the
nucleus; and thus, the same segmentation mask that was used for identification of the nucleus was used for cleaved PARP based measurements (Panels
C and F). In the control well image, no cells were cleaved PARP positive. In the treated well image, most cells showed some level of cleaved PARP
signal but some cells (ROIs 105 and 109) were cleaved PARP negative. Cells that were cleaved PARP positive but not identified with ROIs were nontransfected cells that were excluded from the analysis.
mitochondrial morphology occurs within the first hour of
staurosporine treatment. Similarly, nuclear condensation, a
hallmark of apoptosis, can be detected visually (Panels G – I)
and quantitated (Panel K). These two effects show a similar
trend and time response.
Cells positive for cleaved PARP were virtually undetectable in
control cells (Figure 7, Panel A), but become more numerous
and brighter in intensity over time (Panels B and C). The timedependent increase in the number of cleaved PARP positive
cells is shown in Panel L (% of cells within the wells that were
positive). Quantitation of the increase in average intensity of
the cleaved PARP labeling in positive cells over time is shown in
Panel M.
Apoptosis is a cascade of cellular events, some of which may
be detected earlier than others. Data analysis showed that
significant changes in cytoplasmic (mitochondrial) and nuclear
morphology (38% and 27% loss in size, respectively) occurred
within 1 h of staurosporine treatment. The percentage of cells
positive for cleaved PARP was a slower response with only a
7.5% increase over controls at 1 h and a 50% increase by 3 h.
Detection limits of the probes measured may also influence how
thoroughly cellular responses are measured.
These experiments demonstrate that the Red FP-Mitochondrion
vector is well suited for either live-cell or multiplexed endpoint
analysis of cellular apoptotic events. The results also highlight
that analysis of multiparametric assay data can yield a more
thorough understanding of the underlying biological response.
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SUMMARY
The Green FP and Red FP organelle markers are well suited
for use in high-content and other imaging applications. The
expression and correct localization of the markers were verified
in both transient and stably transfected cells. In live cells, the
probes are useful for kinetic assays alone or in combination
with suitable live-cell dyes. While some fluorescence intensity
was lost upon fixation and permeabilization, the FPs are
still beneficial reagents for endpoint, fixed-cell assays as
demonstrated by the Red FP-Mitochondrion vector in the
apoptosis assay multiplexed with both a dye and an antibody.
In both the live-cell and endpoint apoptosis assays, the results
followed the same trends. This indicates that live-cell assays
can be used to define time points and other parameters for
endpoint assays, and that fixation and permeabilization
procedures do not compromise the utility of FP vectors for
multiplexed endpoint assays. In addition to the apoptosis assay
demonstrated, other assay model systems are possible using one
or more of the eight FP organelle and cytoskeletal biomarkers
available in this collection. In particular, since these markers are
ubiquitously expressed, they can provide very sensitive detection
methods for cellular processes. Availability of these vectors
enables the development of informative cell-based assays for
industrial and academic endeavors.
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Bioimaging Application Note – FP Organelle Vectors
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