Ten ways to accelerate your research with benchtop flow cytometry

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Ten ways to accelerate your research with bench top flow cytometry Jason Whalley Product 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.

description

Analyze 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.

Transcript of Ten ways to accelerate your research with benchtop flow cytometry

Page 1: 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.

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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.

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#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.

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#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.

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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