Ten ways to accelerate your research with benchtop flow cytometry
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Transcript of Ten ways to accelerate your research with benchtop flow cytometry
Ten ways to accelerate your research with bench top flow cytometry
Jason WhalleyProduct Manager Millipore Corporation
Jason Whalley integrates
development and marketing
for the guava bench top flow
cytometers, assay kits, and
analysis software packages at
Millipore, all described at
www.millipore.com/guava10.
2
guava
TM
Normal or neoplastic?
Diploid or aneuploid?
Differentiated or pluripotent?
INtroDuCtIoNAnalyze any cell faster and more
meaningfully with flow cytometry.
Delivering richer, more predictive,
and more quantitative data
compared to other methods of
cell analysis, flow cytometry
processes one cell at a time and
measures multiple proteins per cell
simultaneously. However, the high
costs of instrumentation, extensive
assay development, optimization
and overall expertise required have
limited the use of flow cytometry to
large core facilities and associated
institutions.
the recent development of
bench top flow cytometry grants
anyone access to the power of
flow cytometry, regardless of
expertise, budget, space restrictions
or access to a core facility. Guava
technologies, which recently became
part of Millipore, developed the first
commercially available microcapillary
flow cytometer. this new, compact,
affordable technology helped to
launch the popularity of bench top
flow cytometry. today, the guava®
platform also integrates optimized
assay kits, software and service for
the first end-to-end bench top flow
cytometry solution.
though they are easy to
maintain and operate, guava flow
cytometers span a broadening range
of sophistication, from two-color
detection of single samples, to six-
color detection of multiple tubes or
96-well plates. the newest guava
instrument, the guava easyCyte™
8Ht flow cytometer, measures
up to eight cellular parameters
simultaneously.
Does this sound like the
perfect platform for answering your
research questions? Here are ten
ways to accelerate your discoveries
using bench top flow cytometry.
3
#1: receive instant feedback from colleagues as you acquire cell analysis data right in your lab.
Figure 1. Corroborating biochemistry with whole cell analysisBench top flow cytometry researchers at Millipore Corporation used the FlowCellect™ Stem Cell Characterization Kit to define changes in protein expression during cell dif-ferentiation. Differentiation of human embry-onic stem cells (black population) into neural progenitor cells (white population) results in downregulation of oct4 and SSEA4, as has been also observed biochemically.
Your labmate is performing
quantitative PCr to detect
transcripts encoding proteins
including those you’ve tagged in
your cells. Instead of sitting in an
isolated core lab, pondering the
regulatory mechanism behind your
expression patterns, you could be
sharing ideas with your colleague
just as her PCr curves are appearing
on the thermocycler. If her results
inspire you to modify your flow
cytometry protocol, the opportunity
is at your fingertips.
today’s biological research
questions are more complex than
ever, and forward progress requires
collaborative synergy. Don’t waste
time traveling across buildings,
campuses, or even cities (as some
scientists do!) to reach a shared
cytometer, especially with time- or
temperature-sensitive samples.
With a bench top flow cytometer,
both you and your colleague could
be writing a paper by the end of the
week.
10e0 10e1 10e2 10e3 10e4
hOCT4-Alexa 488
120
90
60
30
0
Coun
t
10e0 10e1 10e2 10e3 10e4
SSEA4-PE
200
150
100
50
0
Coun
t
#2: Spend less time waiting for instrument maintenance or managing waste.
the patented microcapillary
technology allows guava systems
to operate without sheath fluid,
generate less waste and easily fit
on any bench top. Additionally, they
also have lower operating costs,
and are easier to set up and run
than traditional flow cytometers.
All guava flow cytometers feature
daily quality control checks, have
an automated cleaning routine, and
have self-aligning, user-replaceable
flow cells, so you don’t have to wait
for a service technician to fix a clog
or make minor adjustments.
4
#3: train yourself to prepare samples, acquire, and analyze in less than a day.
#4: Get perfect data with the best cell counts and viability tests before every experiment.
the commercialization of turnkey
assay kits and optimized analysis
software makes acquiring flow
cytometry data easy, even if you
have no prior experience. Millipore’s
expanding portfolio of reagent kits
is validated specifically for use
on guava flow cytometers. over
20 of these FlowCellect assay
kits are available for measuring
chemokine receptor levels, stem
cell phenotypes, signaling, cell
growth and toxicity, apoptosis, cell
cycle progression, cell counting
and viability. Just bring your cells,
because all necessary reagents
are not only included, but are also
optimized. Additionally, antibodies
within FlowCellect kits have been
validated by Western blot and
immunocytochemistry, so that you
can be sure your data have true
biological significance.
You observed apparent decrease
in gene expression upon cell
differentiation, only to discover that
your differentiated cell population
was actually apoptotic! Precise,
accurate cell counts provide more
consistency for experiments
that depend upon accurate
determination of viable cells for
success, such as ELISAs, cell-based
fluorescence assays, or cytokine
production assays. All too often,
hematocytometers or other cell
counting methods do not provide
accurate measurements of cell
viability, or you are forced to use
expensive reference beads.
the ViaCount® flow cytometry
assay kit (Millipore) provides
direct, absolute cell counts and
differentiates between viable,
apoptotic, and dead cells, right
on your bench top. With accurate
cell counts, your experiments will
provide much more meaningful
results.
Additional flow cytometry
assay kits can distinguish different
stages of apoptosis. For example,
in the early stages of apoptosis,
phosphatidylserine molecules move
to the outer leaflet of the plasma
membrane, and can then bind
Annexin V protein. the Guava Nexin®
Assay Kit (Millipore) treats cells
with the AAD dye, which permeates
Viability Stain
Nuc
leat
ed C
ells
Live
Apoptotic
Dead
Annexin V
Via
bilit
y Sta
in
Live
Apoptotic
Dead
Figure 2.
Annexin V
Via
bilit
y Sta
in
Live
Apoptotic
Dead
Viability Stain
Nuc
leat
ed C
ells
Live
Apoptotic
Dead
late-stage apoptotic or dead cells,
in addition to labeled Annexin V.
the ViaCount Assay Kit, with the
Guava Nexin Assay Kit, unequivocally
identifies early and late apoptotic
cells in a mixed population. the
right panel of Figure 2 shows that
the intermediate staining (blue)
cells display high levels of Annexin
V binding, confirming that they are
apoptotic.
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Figure 3. Chemokine Receptor Quantification
10e0 10e1 10e2 10e3 10e4
M1
6%MFI=8.54 98%98%
MFI=588MFI=588
96%96%MFI=273MFI=273
200
150
100
50
0
Cou
nt
Yellow Fluorescence (YLW-HLog)
Wild Type “negative” control cells(< 20,000 receptors/cell; MFI=8.54)
CXCR4/Chem-1 “positive” control cells(380,000 receptors/cell; MFI=588)
Jurkat T cells (Native cell line)(MFI=273, equivalent to approx. 176,320 receptors/cell)
#7: Quickly test new cellular pathways outside your field of expertise.
#6: Skip time-consuming microscopy or radioligand binding assays.
#5: Gain speed and accuracy with lower required cell numbers.
Even if your expertise is in cell
cycle research, using an integrated
bench top flow cytometry system,
such as the combination of guava
flow cytometers, FlowCellect kits,
and guavaSuite™ software, makes
it easy to test your research model
for receptor signaling, apoptosis,
or other unfamiliar pathways. the
assay kits take the guesswork out
of sample preparation, giving you
the conditions and controls that
work best.
Flow cytometry assay kits have a
clear advantage over traditional,
laborious microscopy and radioligand
binding assays for quantifying
cell surface proteins such as
chemokine receptors. regulating
leukocyte activation and trafficking,
chemokine receptors govern HIV
infection, rheumatoid arthritis,
diabetes, immune disorders and
inflammation. the levels of these
receptors on the cell surface
indicate normal or diseased states.
twelve FlowCellect assay kits
measure expression of twelve
different chemokine receptors.
Each assay includes a receptor-
Don’t spend several days
accumulating enough sample for
biochemical assays. through use of
the guava microcapillary technology,
you can achieve accurate,
reproducible, physiologically relevant
cell analysis with 500,000 cells in
as little as 200 μL sample volume.
Instead of spending hours and
days in the tissue culture hood and
specific antibody validated and
optimized for guava instruments,
labeled secondary antibody, and
control cell lines either expressing
or not expressing the recombinant
receptor at known levels, for easy,
fast receptor quantitation.
In Figure 3, chemokine receptor
CXCr4 was measured in Jurkat t
cells using the CXCr4 FlowCellect
kit. Comparing the fluorescence
signal of the Jurkat cells to the
CXCr4 stable cells and null cells
revealed the number of receptors
per cell, which matched the number
previously calculated by radioligand
binding.
risking drifting phenotypes, use
guava bench top flow cytometry to
get an accurate, real-time snapshot
of your cells.
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#8: Analyze meaningful data and biological significance faster for each assay.
Millipore’s guavaSuite assay-specific
software modules help you get to
the most meaningful data more
quickly, with friendly, step-by-step
guides incorporated into the user
interface. optimized for each assay,
the software modules display only
the pre-set plots and statistics
you need. Each software module
starts with one-click acquisition
and analysis, then exports results
to a database without additional
user intervention. this integrated,
automated process makes your cell
analysis both easier and more ac-
curate.
Guava systems also include
InCyte™ Software, the first analysis
package designed specifically to
give every user the power to draw
conclusions about the biological
significance of data. Its intuitive,
easy-to-use interface displays and
compares up to eight data sets at
the same time.
Drag and drop gating – from one plot to another
View up to 11 plots at once
real time plot adjustments
organize acquired data sets and select individual wells for display
Quickly link to and review previously analyzed data
Easily create analysis templates
Combine groups of data and analysis templates to construct heat maps or EC50 curves
Slider bars set cut-offs or threshold values for each experimental sector
Heat map shows values across an entire plate
Figure 4. InCyte Software is both intuitive and powerful.
InCyte enables you to analyze
entire plates of data in less time
than it takes to analyze a single
sample. Most importantly, com-
parative results are displayed at
the experiment level rather than
on an individual well/sample basis.
this software is especially use-
ful for interpreting the results of
sirNA screens, apoptosis/cell cycle
compound screening or other high-
throughput, high content, cell-based
assays.
www.millipore.com/guava10
Millipore, Guava, Viacount and Guava Nexin are registered trademarks of Millipore Corporation. guava easyCyte, FlowCellect, InCyte, guavaSuite, the “M” mark, and Advancing Life Science together are trademarks of Millipore Corporation.LS-SBu-09-02232 08/09 Printed in u.S.A.
© 2009 Millipore Corporation, Billerica, MA 01821 All rights reserved.
WhAT’S nExT?
Not only does bench top flow
cytometry give you speed, but, more
importantly, it enables flexibility,
spontaneity, and collaboration. It’s a
combination of these benefits that
will help you truly change the world
with your next discovery.
#9: Speed up iterative flow cytometry-based analyses.
#10: turn around instant results for translational, clinical or environmental research.
If you’re trying to select a stable,
fluorescent reporter cell line, or
undertaking a large-scale, pooled
sirNA screen, you need to perform
multiple rounds of flow cytometry,
each round’s protocol dependent on
the results of the last. With a flow
cytometer on your bench top, you
can start the next round of experi-
ments just as soon as you finish the
last one. No guessing, waiting, or,
worst of all, worrying.
When your lab bench is closely tied
to a patient’s bedside, you need ac-
cess to powerful cell analysis within
the clinical research or translational
research laboratory. With bench top
flow cytometry at hand, 24 hours a
day, you can correlate flow cytome-
try data with conventional radiology
or histopathology results, enabling
better fertility treatments, and more
accurate therapeutic evaluation.
In the environmental research
sector, you may be conducting re-
search in extreme climates, far from
any shared flow cytometry facility,
perhaps on microbes that are re-
sistant to culturing. Bench top flow
cytometry provides the opportunity
to analyze microbes on the spot,
testing novel enzymatic activities, or
assessing their response to com-
pounds.
to learn more about integrating
a flow cytometry workflow into
your research program, basic and
advanced flow cytometry principles,
and details of the guava bench top
flow cytometry platform, visit
http://www.millipore.com/guava10