No. 45 –  2016
>
>
Pipetting with a Smile:
How to Enjoy Pipetting
(BN 45) JUNE 2016
PAGE 1
Faster Isolation of PBMCs Using Ficoll-Paque™ PLUS
in the Eppendorf Centrifuge 5920 R
>Handling of Difficult Liquids Made Easy
>New Centrifuge 5920 R with 4 Liter Capacity
>Eppendorf Sterilize-in-Place Bioprocess Systems
Application Notes
Faster Isolation of PBMCs Using Ficoll-Paque® PLUS in the Eppendorf Centrifuge 5920 R · ­
Faster and More Reproducible Cell Viability Assays with the Eppendorf epMotion® 96 · etc.
NICOLE SEELIGMÜLLER, EPPENDORF AG, HAMBURG, GERMANY
Introduction
Human blood consists of equal parts
of blood plasma and blood cells. These
include erythrocytes (red blood cells),
leukocyctes (white blood cells), and
thrombocytes (platelets). Leukocytes
are further subdivided into different
cell types.
These include, for example, lymphocytes
and monocytes, which (in co-operation
with other cells) form the basis of the
innate immune system and which, owing
to their simple nucleus, are referred to
as peripheral blood mononuclear cells
(PBMCs). The term lymphocyte encompasses two major classes, B lymphocytes
and T lymphocytes. B lymphocytes are
responsible for antibody production,
whereas T lymphocytes produce signal
molecules which will finally lead to the
removal of diseased or foreign cells.
PBMCs play an important role in the
areas of infection diagnostics as well
as in clinical research [1].
PBMCs are isolated from buffy coats
(whole blood concentrates without
serum). Centrifugation using FicollPaque™ PLUS (density: 1.007 g/mL)
allows separation of PBMCs from other
blood components: erythrocytes, granulocytes, and dead cells which, due to
their higher density, will pass through
the Ficoll layer, whereas lymphocytes
and monocytes will accumulate at the
interphase between plasma and Ficoll
gradient (Fig. 1), [2,3].
One prerequisite for a clean PBMC
isolation with a maximum yield of living
cells is the formation of a clear interphase. For this reason, the procedure
must be carried out with the least
vibration possible.
Usually, mixing of the phases can only
be avoided by centrifugation with the
rotor brake deactivated [4], which constitutes an extremely time-consuming
step within this application.
This study shows that the new 4 liter
benchtop Centrifuge 5920 R not only
satisfies the high demands of this
method, but that thanks to the option
of selecting individual ramps, the user
will enjoy considerable time savings.
Materials and methods
Ficoll-Paque PLUS density gradient
centrifugation
1. Fill Eppendorf Conical Tubes 50 mL
with 15 mL Ficoll-Paque PLUS
(GE Healthcare®) each.
2. Using the slowest pipetting speed
available, overlay the Ficoll with
15 mL of the blood/PBS mixture.
3. Centrifuge the samples for 30 min
in the swing-bucket rotor at 400 x g,
20 °C, at ramp* 0/0 or ramp 3/3,
respectively, while selecting the
setting “at set rpm”.
*Possible ramp settings Centrifuge 5920 R: deactivated
acceleration or deceleration, respectively (0/0) to fastest
acceleration/brake (9/9)
Purification of lymphocytes
1. Carefully remove 2/3 of the upper
layer (plasma).
2. Aspirate as much of the PBMC layer
as possible in the smallest volume
possible and transfer to a fresh tube.
3. Wash PBMCs several times in PBS
and resuspend in suitable cell culture medium.
Viability test and determination of yield
1. Dilute cells 1:1 in trypan blue and
count.
2. Determine viability and yield.
Detailed information about materials
and methods is available in Application
Note 372 [5].
Results and discussion
In order to evaluate the quality of separation, it was determined whether a
defined interphase with clearly delineated phase transitions was visible. Fig. 2
(A–D) shows the results obtained from
the density gradient centrifugations
carried out in the Centrifuge 5920 R
using different swing-bucket rotors.
For each rotor, the acceleration and
deceleration rates 0/0 and 3/3 were
tested. It is evident that optimal separation of blood components was achieved
in all cases, and that vortex effects
during deceleration of the rotor could
be completely avoided (see negative
example Fig. 2E).
Plasma
PBMC
Ficoll
Granulocytes
Erythrocytes
A
B
C
D
Fig. 2: Results obtained after density gradient centrifugation in the Centrifuge 5920 R
Fig. 1: Density gradient obtained from buffy coat using
Ficoll-Paque PLUS (schematic)
A) Rotor S-4x1000 with high-capacity bucket (ramp 0/0) B) Rotor S-4x1000 with high-capacity bucket (ramp 3/3)
C) Rotor S-4xUniversal-Large (ramp 0/0) D) Rotor S-4xUniversal-Large (ramp 3/3)
E) Negative example: cloudy interphase, turbid plasma-/Ficoll phase (different centrifuge)
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
E
2
EDITORIAL · DEAR READER
Imprint
Editorial team
Berrit Hoff (Editor-in-Chief), Axel Jahns,
Jochen Müller-Ibeler, Tanja Musiol,
Natascha Weiß
Publisher
Eppendorf AG, Barkhausenweg 1,
22339 Hamburg, Germany
Telephone: (+49) 40-53801-0
Fax: (+49) 40-53801-556
E-mail: [email protected]
Web: www.eppendorf.com
We welcome all readers’ articles for this
Dear Reader!
publication. However, no responsibility is
accepted for unsolicited manuscripts.
Important note
The new products described may be
In 1962, Eppendorf launched a revolutionary invention: the modern microliter pipette.
Since that time, the world of pipetting has continually evolved, but so has our product
portfolio.
>
With pipetting solutions for every taste and demand, as well as a broad spectrum of
support and training opportunities, we aim to make your daily pipetting routine as
pleasant as possible, as you will discover on pages 4 – 5. Here we will also introduce to
you our new internet platform www.eppendorf.com/pipetting, where you will find
­numerous tips, tricks, and stories all about pipetting. Perhaps you will even recognize
yourself in one of our new cartoons and start your next pipetting series with a smile!
The topic of liquid handling will recur several times throughout this issue: in the contribution on the handling of difficult liquids (p. 6), in the article featuring the high chemical
resistance of our pipette controller Easypet® 3 (p. 10), as well as in the study on the influence of different liquid handling instruments on cell assay performance (Application
Note p. 3 – 4).
Remember part 1 of our Application Note series “The Tip of the Iceberg”? For the
­sequel, the authors have scrutinized the openings of different pipette tips using
­microscopy – with impressive results (Application Note p. 5 – 6).
In this issue, we proudly present you the refrigerated Centrifuge 5920 R, a true miracle
of performance and space. With similar dimensions as a 3 liter centrifuge, it provides a
capacity of up to 4 x 1,000 mL or 52 x 50 mL conical tubes. The Centrifuge 5920 R is
the perfect combination of user-friendliness and precision (p. 8 and Application Note
p. 1 – 2).
The family of Eppendorf Tubes® 5.0 mL continues to grow! The new screw cap tube
5.0 mL was developed for certain applications in the medium volume range, where a
screw cap is beneficial to either experiment or sample handling. Learn more about the
new “Eppi” on page 11.
We wish you an exciting read!
Your BioNews team
launched at different times in various
countries. Please contact your local
Eppendorf organization or distributor
for details.
Technical specifications subject to change.
Errors and omissions excepted.
All rights reserved,
including graphics and images.
© Copyright Eppendorf AG, June 2016.
Carbon neutrally printed in Germany.
>
CONTENTS
4
IN THE SPOTLIGHT
STRAIGHT FROM THE LAB
INNOVATION
>
8
Pipetting – with a Smile
10
4 – 5
Proper Handling of Cells in the Lab (I)
7
State-of-the-Art DNA Measurement: Efficient, Fast, and Reproducible
9
Robust and Reliable: Easypet® 3
10
Holistic Approach to Ergonomics
12
Eppendorf Sterilize-in-Place (SIP) Bioprocess Systems
13
Handling of Difficult Liquids Made Easy
6
Centrifuge 5920 R: New Benchmark in Capacity and Performance
8
Eppendorf Tubes® 5.0 mL – Now Also Available with Screw Cap
CLOSE-UP
NEWS / TIPS
SERVICE
(BN 45) JUNE 2016
PAGE 1
Faster Isolation of PBMCs Using Ficoll-Paque™ PLUS
in the Eppendorf Centrifuge 5920 R
NICOLE SEELIGMÜLLER, EPPENDORF AG, HAMBURG, GERMANY
Introduction
Human blood consists of equal parts
of blood plasma and blood cells. These
include erythrocytes (red blood cells),
leukocyctes (white blood cells), and
thrombocytes (platelets). Leukocytes
are further subdivided into different
cell types.
These include, for example, lymphocytes
and monocytes, which (in co-operation
with other cells) form the basis of the
innate immune system and which, owing
to their simple nucleus, are referred to
as peripheral blood mononuclear cells
(PBMCs). The term lymphocyte encompasses two major classes, B lymphocytes
and T lymphocytes. B lymphocytes are
responsible for antibody production,
whereas T lymphocytes produce signal
molecules which will finally lead to the
removal of diseased or foreign cells.
PBMCs play an important role in the
areas of infection diagnostics as well
as in clinical research [1].
PBMCs are isolated from buffy coats
(whole blood concentrates without
serum). Centrifugation using FicollPaque™ PLUS (density: 1.007 g/mL)
allows separation of PBMCs from other
blood components: erythrocytes, granulocytes, and dead cells which, due to
their higher density, will pass through
the Ficoll layer, whereas lymphocytes
and monocytes will accumulate at the
interphase between plasma and Ficoll
gradient (Fig. 1), [2,3].
One prerequisite for a clean PBMC
isolation with a maximum yield of living
cells is the formation of a clear interphase. For this reason, the procedure
must be carried out with the least
vibration possible.
Usually, mixing of the phases can only
be avoided by centrifugation with the
rotor brake deactivated [4], which constitutes an extremely time-consuming
step within this application.
This study shows that the new 4 liter
benchtop Centrifuge 5920 R not only
satisfies the high demands of this
method, but that thanks to the option
of selecting individual ramps, the user
will enjoy considerable time savings.
Materials and methods
Ficoll-Paque PLUS density gradient
centrifugation
1. Fill Eppendorf Conical Tubes 50 mL
with 15 mL Ficoll-Paque PLUS
(GE Healthcare®) each.
2. Using the slowest pipetting speed
available, overlay the Ficoll with
15 mL of the blood/PBS mixture.
3. Centrifuge the samples for 30 min
in the swing-bucket rotor at 400 x g,
20 °C, at ramp* 0/0 or ramp 3/3,
respectively, while selecting the
setting “at set rpm”.
*Possible ramp settings Centrifuge 5920 R: deactivated
acceleration or deceleration, respectively (0/0) to fastest
acceleration/brake (9/9)
Purification of lymphocytes
1. Carefully remove 2/3 of the upper
layer (plasma).
2. Aspirate as much of the PBMC layer
as possible in the smallest volume
possible and transfer to a fresh tube.
3. Wash PBMCs several times in PBS
and resuspend in suitable cell culture medium.
Viability test and determination of yield
1. Dilute cells 1:1 in trypan blue and
count.
2. Determine viability and yield.
Detailed information about materials
and methods is available in Application
Note 372 [5].
Results and discussion
In order to evaluate the quality of separation, it was determined whether a
defined interphase with clearly delineated phase transitions was visible. Fig. 2
(A–D) shows the results obtained from
the density gradient centrifugations
carried out in the Centrifuge 5920 R
using different swing-bucket rotors.
For each rotor, the acceleration and
deceleration rates 0/0 and 3/3 were
tested. It is evident that optimal separation of blood components was achieved
in all cases, and that vortex effects
during deceleration of the rotor could
be completely avoided (see negative
example Fig. 2E).
11
ConvexAccess : Improved Handling, Increased Safety
7
Increase Your Pipetting Knowledge!
5
™
Supporting You: Maintenance and Certification Services
10
Eppendorf Services
12
Eppendorf Prize Winners 2015/2016 Shigeki Watanabe & Adrian Liston
14
Trademarks Information
14
Win a Multichannel Pipette!
15
NICOLE SEELIGMÜLLER
Faster Isolation of PBMCs Using Ficoll-Paque® PLUS
in the Eppendorf Centrifuge 5920 R
1 – 2
ERIC GANCAREK, MAUD BRASSEUR, MURIEL ART, HANAË A. HENKE
Faster and More Reproducible Cell Viability Assays
with the Eppendorf epMotion® 96
3 – 4
ULRIKE GAST, LAURA KOCH
The Tip of the Iceberg: How Pipette Tips Influence Results.
Part 2: Perfect Geometry Makes a Difference
5 – 6
Plasma
PBMC
MAREIKE PANZ, JOHANNA PRIEB, INES HARTMANN
Ficoll
Granulocytes
Erythrocytes
A
B
C
D
Fig. 2: Results obtained after density gradient centrifugation in the Centrifuge 5920 R
Fig. 1: Density gradient obtained from buffy coat using
Ficoll-Paque PLUS (schematic)
A) Rotor S-4x1000 with high-capacity bucket (ramp 0/0) B) Rotor S-4x1000 with high-capacity bucket (ramp 3/3)
C) Rotor S-4xUniversal-Large (ramp 0/0) D) Rotor S-4xUniversal-Large (ramp 3/3)
E) Negative example: cloudy interphase, turbid plasma-/Ficoll phase (different centrifuge)
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
E
Improved Automated Single Cell Clone Analysis with the
SynenTec Cellavista® System and Eppendorf Cell Culture Plates
3
7 – 8
>
4
IN THE SPOTLIGHT · PIPETTING – WITH A SMILE
KAY KÖRNER, EPPENDORF AG
Pipetting – with a Smile
In the year 1962, Eppendorf launched a revolutionary invention to the market: the modern microliter pipette. Since
that time, the world of pipetting has continually evolved. Previously unattainable demands on ergonomics and precision have become the new standard. Understandably, the expectations of modern science on the reproducibility
of results have risen in parallel. As a variable which is difficult to control, the human factor can influence results
significantly. This is where modern pipetting systems must exert their leverage – with a focus on easy handling.
­ ppendorf invests much time and brainE
power in the development of products
which allow the user to enter the state of
flow: lightweight pipettes, optimized tip
fit, and high-contrast plate labeling. If
you take a closer look or speak with one
of our team members, you will discover
a multitude of product features which
can ease your life significantly.
>
Occasionally, potential problems may
hide in the smallest details, invisible to
the naked eye. For example, our laboratory had problems obtaining reproducible
results when using pipette tips by other
manufacturers. The Application Note on
p. 5 – 6 in this issue “The Tip of the Iceberg: How Pipette Tips Influence Results.
Part 2: Perfect Geometry Makes a Difference” provides more information on this
topic.
Really good pipetting is an acquired skill
You too can pipette with a smile!
Do you enjoy pipetting?
The responses to this question range
from “yes” with a smile to an eye-rolling
“no”. As we know from experience, we
enjoy activities when we are proficient,
don’t suffer, and finally achieve success –
and we enjoy a challenge. Psychologists
call this state “flow” – the happy feeling of
complete immersion in or concentration
on a task.
Some experience this state of flow during model construction or while playing
soccer, while others find their happiness
while pipetting: the purposeful striving
to reach the 96th well and the trance-like
state of repeating the same actions. However, blessing and burden are sometimes
closely linked. A decisive factor which
can make the difference between flow
and frustration is also the quality of the
instruments used.
Getting into the flow
A pipette that feels heavy, pipette tips
that don’t fit. A plate on which the labeling is difficult to read. All these shortcomings not only tire the body, but also
the spirit. A feeling of fun and ease is
hard to come by. For these reasons,
Really good pipetting, however, takes more
than really good products. Mastering the
right techniques and acquiring expertise
are essential. This is why Eppendorf,
­beyond developing really good products,
takes on the responsibility of providing
the user with the knowledge required to
reach the state of flow.
For example, in addition to online webinars (see www.eppendorf.com/webinar),
Eppendorf also offers pipetting training
at different sites around the world.
For example, do you recall off hand the
exact density at which reverse pipetting
>
PIPETTING – WITH A SMILE · IN THE SPOTLIGHT
5
Tip
Increase Your
Pipetting
Knowledge!
Exact pipetting is a prerequisite for reliable
results. Pipetting, at first glance, appears to
be a simple action. In fact, the pipetting
results can be influenced by numerous
factors that are listed here from A to Z!
Influencing factors on pipetting results
becomes beneficial? This and other questions will be addressed, not only during
our training sessions, but also on our new
internet platform www.eppendorf.com/
pipetting.
>
On this new platform you will not only
find a multitude of tips and tricks on pipetting. You can also test your pipetting
expertise in a quiz challenge or discover
if you recognize your daily lab routine in
one of our cartoons which may help you
survive the next long pipetting row with
a smile!
Automated pipetting
In order to tame the human factor mentioned at the beginning, solutions for
­automated pipetting are available. The
influence of the human hand as a source
of uncertainty is eliminated to a large extent (in the case of an electronic pipette)
or even entirely (in the case of a liquid
handling workstation). A liquid handling
workstation increases the reproducibility
of pipetting results considerably as factors
such as the pipetting angle, speed, and
other similar parameters are largely kept
constant. Especially since high accuracy
is achieved, liquid handling workstations
are capable of pipetting small volumes
much more reliably than the human
hand. This way, assays may be carried
out using smaller volumes of r­ eagent –
this saves money.
A
Adjustment settings
B
Body temperature and heat transfer
C
Calibration, last
D
Density of liquid
E
Environmental conditions
F
Foaming liquids
G
Geographical height and air pressure
H
Humidity of air
I
Immersion depth
J
Just wait: waiting time after aspiration
K
Knowledge of pipetting techniques
While liquid handling workstations may
hardly induce a state of flow, they will
certainly help save a lot of time for
“flow-worthy” pursuits.
L
Level of pipetting speed – work slowly
M
Maintenance status
N
Ninety degrees – hold pipette vertically
O
Operational vapor pressure
P
Prewetting
Pipetting solutions for every taste
Q
Quality and design of pipette tip
R
Rhythm of pipetting
S
Size of air-cushion (as small as possible)
T
Temperature
U
Under stress: a tired hand does not pipette
accurately
V
Viscosity
W
Wetting behavior of liquid
X
XXL temperature difference between sample and
pipette
Y
Dispense against wall in same angle as the arms
of a Y
Z
Zero patience
With our today’s selection of pipetting
solutions, we are sure to offer the right
product for every taste – whether you
are pipetting with a smile or not. Finding
the right pipette back in 1962, however,
was very easy. There was only one
­microliter pipette, and it was made by
Eppendorf.
Would you like to learn more?
A wide selection of Eppendorf videos on
pipetting can be found at
www.eppendorf.com/
playlist-eppendorf-pipettes
or by entering the QR code!
The automated liquid
handling systems of the
epMotion® series by
Eppendorf are available
in four different formats
and with various­
­upgrading options.
>
6
INNOVATION · HANDLING OF DIFFICULT LIQUIDS MADE EASY
RUDOLF WALCZAK, EPPENDORF AG
Handling of Difficult Liquids
Made Easy
Nearly every life science researcher works with liquids, and viscous or volatile liquids pose a significant challenge.
Handling them correctly requires specialized knowledge as well as the proper tools. Eppendorf offers its customers a broad portfolio of products for liquid handling. The electronic dispensers Multipette® E3 and Multipette E3x
(U.S./CAN: Repeater® E3/Repeater E3x) represent the latest innovations for working with difficult liquids and for
handling long dispensing series.
>
Effortless handling of difficult liquids
Water is the main component of living
cells, and the majority of biological
­processes occur in an aqueous environment. As a consequence, biochemists,
molecular biologists, and researchers in
related disciplines conduct their experiments primarily in aqueous solutions.
A number of options are available for
properly handling difficult liquids. In addition to special pipetting techniques (e.g.
reverse pipetting), specialized pipette
tips may be employed (e.g. epT.I.P.S.®
LoRetention or ep Dualfilter T.I.P.S.®).
Difficult liquids: not the exception
However, liquids are as different as the
people who work with them. As soon as
their physical properties deviate significantly from water or simple salt solutions, liquids pose a special challenge.
Multipette/Repeater E3 and E3x: innovative experts for
difficult liquids and long dispensing series
For example, viscous or especially dense
solutions are difficult to pipette using
air-cushion pipettes. Volatile liquids, on
the other hand, will drip from the pipette
tip due to their high vapor pressure. Solutions that contain detergents or proteins tend to foam, which bears the risk
of sample contamination. Finally, even
cold or warm liquids (relative to ambient
temperature) may be classified as difficult.
Due to the difference in temperature, the
air-cushion above the liquid will expand
or contract, which can lead to systematic
pipetting errors. Furthermore, the formation of aerosols by warm liquids may
contribute to corrosion of the pipette.
Difficult liquids are more common than
generally appreciated, and they play an
important role in a number of different
areas, for example in diagnostic laboratories as well as in the chemical and
­cosmetics industries.
Common difficult liquids
Property
Examples
High viscosity
Glycerol, blood, mineral oil
Volatile
Ethanol, acetone, chloroform
High density
Sulfuric acid, cesium chloride solutions
Foaming
Tween™ 20, Triton™ X-100, protein solutions
Cold
Enzyme solutions, cooled buffers
Warm
Cell culture medium
The safest solution, however, is using a
positive-displacement system which dispenses the liquid directly, without the
need for an air-cushion. For this purpose,
Eppendorf has developed the Multipette
(Repeater)/Combitips® system.
All Multipettes/Repeaters enable precise
and fast dispensing of a broad range of
liquids. The Multipettes/Repeaters E3
and E3x (with their different dispensing
modes) offer the additional benefits of
an electronic instrument. They make a
large number of liquid handling applications accessible and support increased
throughput. This way, working with difficult liquids becomes really easy.
More information
Additional information is available
in the product brochure or at
www.eppendorf.com/
multipette-system.
Multipette ® E3 and E3x (U.S./Can: Repeater ® E3 and E3x) •
Ref. no. 290
>
PROPER HANDLING OF CELLS IN THE LAB (I) · STRAIGHT FROM THE LAB
7
TANJA MUSIOL, EPPENDORF AG
Proper Handling of
Cells in the Lab (I)
Cell culture work, which includes seeding, cultivation, and analysis, holds a
Close-up
ConvexAccess™:
Improved Handling,
Increased Safety
Cell culture flasks are the preferred
special place in the daily laboratory routine. Whenever cells are concerned,
solution for routine applications involving
experimental conditions are as different as the cells themselves.
long-term cultivation or large-scale
expansion of cells. Optimal protection of
cells from contamination was one of the
key requirements in developing the
Eppendorf Cell Culture Flasks.
To meet these demands, Eppendorf not
only developed an exceptionally efficient
>
Sources of contamination
Prevention of contamination
No matter whether primary cells, stem
cells, or cell lines are the focus of one’s
work, the demands on cell handling can
be quite diverse, depending on the specific cell type as well as the nature of the
experiment.
The multitude of possible contamination
sources points out the limited usefulness
of antibiotics added to the culture. While
a large proportion of bacteria may be
combatted in this manner, contamination
of a different nature may be not. This fact
harbors the additional threat of undetected low level contaminations with resistant strains which are difficult to detect. Nonetheless, the contamination
persists within the culture and therefore
poses a danger to the entire cell culture
laboratory. In order to prevent carry-over
contamination to other cultures, it is imperative that equipment be thoroughly
cleaned, or exchanged, after each cell line.
To summarize, sterility is still the overarching principle wherever cell culture is
concerned.
That being said, a few general facts need
to be considered when working with cells,
most importantly those concerning the
prevention of contaminations. However,
there are obviously more sources of contamination than spring to mind. Not only
bacteria, but also mycoplasmas, yeasts,
and cells from other cultures pose a
­potential contamination threat, where
especially the latter often result from
carry-over contamination.
More information
Basic tips on contamination prevention
are available at
www.eppendorf.com/cellbiology.
In addition, we recommend the webinar
“Preventing Contamination in Cell
­Culture Labs” (which may be found at
www.eppendorf.com/webinar in the
category “Recorded”).
air filter technology. We also created the
ConvexAccess-shape of the flask neck
which is unique on the market. It greatly
facilitates the access to the entire growth
surface. Cell seeding and media change
have now become more comfortable,
reliable, and safe.
Product features Eppendorf Cell Culture
Flasks
>> ConvexAccess geometry for easier and
safe cell treatment
>> Optimized gas exchange and protection
against contamination by high efficiency
air filter technology
>> Anti-rolling cap which can be stably
positioned on its side facilitating
cultivation steps and minimizing the
contamination risk
>> In-line pressure testing for each flask
guarantees leakage-free flasks for
maximum workflow safety
>> Three sizes: T–25, T–75, and T–175, with
TC-treated or non-treated surfaces, filter
caps or plug-seal caps
More information at
ccc.eppendorf.com/flasks.php
Eppendorf Cell Culture Consumables • Ref. no. 270
>
8
INNOVATION · CENTRIFUGE 5920 R: NEW BENCHMARK IN CAPACITY AND PERFORMANCE
PETER SCHREINER, EPPENDORF AG
Centrifuge 5920 R: New Benchmark
in Capacity and Performance
The new refrigerated Centrifuge 5920 R by Eppendorf sets new standards for benchtop centrifuges. With its
tremendous capacity of up to 4 x 1,000 mL or 52 x 50 mL conical tubes it is the ideal instrument to handle highthroughput applications. This absolute eye-catcher was awarded the Good Design Award 2015 of the Chicago
Athenaeum Museum of Architecture and Design for its compact and ergonomic design.
temperature, and time only have to be saved once to make them
easily accessible via program key. This saves time!
Reliable temperature management
>
As awarded by The Chicago
Athenaeum: Museum of
Architecture and Design.
The state-of-the-art refrigeration system with modern temperature management ensures that the selected temperature is
maintained as accurately as possible and that temperaturesensitive samples are safely centrifuged. The FastTemp pro®
function allows programming of date and time for automatic
pre-cooling.
Versatile and innovative
With its extensive accessories-program for rotors, adapters,
and aerosol-tight caps and lids, the Centrifuge 5920 R is exceptionally versatile. The new universal buckets are especially
innovative as they hold both tubes and plates (Fig. 2).
Fig. 1. Centrifuge 5920 R: similar dimensions to those of a regular refrigerated 3 liter centrifuge,
but with a capacity of 4 liters
Extraordinary capacity
Customers with high-throughput applications will be delighted:
The new refrigerated Centrifuge 5920 R (Fig. 1) offers almost
double the tube capacity of the popular Centrifuge 5810 R.
The capacity has been increased from 28 x 50 mL to 52 x 50 mL
for conical tubes and from 92 x 5 mL to 196 x 5 mL for blood
collection tubes. The maximum volume has increased as well.
The 5920 R holds four 1 liter Nalgene® bottles, setting a new
standard for benchtop centrifuges.
User-friendliness and precision: the perfect combination
The Centrifuge 5920 R is equipped with an advanced operating
system featuring a user-friendly interface for intuitive and comfortable handling. At the same time, the operating system was
designed to enable accurate and reproducible results. Five
­program keys allow fast and easy access to routine programs.
For frequently used applications, parameters such as speed,
Fig. 2: Swing-bucket rotor
S-4xUniversal-Large with
innovative universal buckets
This previously required separate rotor buckets, and sometimes even separate rotors. With this new product solution,
one rotor will suffice. This saves money!
More information at www.eppendorf.com/together
Centrifuge 5920 R • Ref. no. 289
>
(BN 45) JUNE 2016 PAGE 1
Faster Isolation of PBMCs Using Ficoll-Paque® PLUS
in the Eppendorf Centrifuge 5920 R
NICOLE SEELIGMÜLLER, EPPENDORF AG, HAMBURG, GERMANY
Introduction
Human blood consists of equal parts
of blood plasma and blood cells. These
include erythrocytes (red blood cells),
leukocyctes (white blood cells), and
thrombocytes (platelets). Leukocytes
are further subdivided into different
cell types.
>
These include, for example, lymphocytes
and monocytes, which (in co-operation
with other cells) form the basis of the
innate immune system and which, owing
to their simple nucleus, are referred to
as peripheral blood mononuclear cells
(PBMCs). The term lymphocyte encompasses two major classes, B lymphocytes
and T lymphocytes. B lymphocytes are
responsible for antibody production,
whereas T lymphocytes produce signal
molecules which will finally lead to the
removal of diseased or foreign cells.
PBMCs play an important role in the
areas of infection diagnostics as well
as in clinical research [1].
PBMCs are isolated from buffy coats
(whole blood concentrates without
­serum). Centrifugation using FicollPaque PLUS (density: 1.007 g/mL)
­allows separation of PBMCs from other
blood components: erythrocytes, granulocytes, and dead cells which, due to
their higher density, will pass through
the Ficoll layer, whereas lymphocytes
and monocytes will accumulate at the
interphase between plasma and Ficoll
gradient (Fig. 1), [2,3].
One prerequisite for a clean PBMC
­isolation with a maximum yield of living
cells is the formation of a clear interphase. For this reason, the procedure
must be carried out with the least
­vibration possible.
Usually, mixing of the phases can only
be avoided by centrifugation with the
rotor brake deactivated [4], which constitutes an extremely time-consuming
step within this application.
This study shows that the new 4 liter
benchtop Centrifuge 5920 R* not only
satisfies the high demands of this
method, but that thanks to the option
of selecting individual ramps, the user
will enjoy considerable time savings.
Materials and methods
Ficoll-Paque PLUS density gradient
­centrifugation
1.Fill Eppendorf Conical Tubes 50 mL
with 15 mL Ficoll-Paque PLUS
(GE Healthcare®) each.
2.Using the slowest pipetting speed
available, overlay the Ficoll with
15 mL of the blood/PBS mixture.
3.Centrifuge the samples for 30 min
in the swing-bucket rotor at 400 x g,
20 °C, at ramp** 0/0 or ramp 3/3,
­respectively, while selecting the
­setting “at set rpm”.
**Possible ramp settings Centrifuge 5920 R: deactivated
acceleration or deceleration, respectively (0/0) to fastest
acceleration/brake (9/9)
Purification of lymphocytes
1.Carefully remove 2/3 of the upper
layer (plasma).
2.Aspirate as much of the PBMC layer
as possible in the smallest volume
possible and transfer to a fresh tube.
3.Wash PBMCs several times in PBS
and resuspend in suitable cell culture medium.
Viability test and determination of yield
1.Dilute cells 1:1 in trypan blue and
count.
2. Determine viability and yield.
Detailed information about materials
and methods is available in Application
Note 372 [5].
Results and discussion
In order to evaluate the quality of separation, it was determined whether a
­defined interphase with clearly delineated phase transitions was visible.
Fig. 2 (A–D) shows the results obtained
from density gradient centrifugation
carried out in the Centrifuge 5920 R
­using d
­ ifferent swing-bucket rotors.
For each rotor, the acceleration and
­deceleration rates 0/0 and 3/3 were
tested. It is evident that optimal separation of blood components was achieved
in all cases, and that vortex effects
­during deceleration of the rotor could
be completely avoided (see negative
example Fig. 2E).
Plasma
PBMC
Ficoll
Granulocytes
Erythrocytes
A
B
C
D
Fig. 2: Results obtained after density gradient centrifugation in the Centrifuge 5920 R
Fig. 1: Density gradient obtained from buffy coat using
Ficoll-Paque PLUS (schematic)
A) Rotor S-4x1000 with high-capacity bucket (ramp 0/0) B) Rotor S-4x1000 with high-capacity bucket (ramp 3/3)
C) Rotor S-4xUniversal-Large (ramp 0/0) D) Rotor S-4xUniversal-Large (ramp 3/3)
E) Negative example: cloudy interphase, turbid plasma-/Ficoll phase (different centrifuge)
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Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
E
>
PAGE 2 (BN 45) JUNE 2016
Faster Isolation of PBMCs Using Ficoll-Paque® PLUS
in the Eppendorf Centrifuge 5920 R
Conclusion
In order to obtain a high yield of viable
PBMCs, Eppendorf recommends the
use of the new Centrifuge 5920 R for
density gradient centrifugation using
Ficoll-Paque PLUS. According to available test results, consistent high quality
of yield can be expected with all available swing-bucket rotors for 50 mL
conical tubes. With its large capacity
and the option of significantly reducing
the time ­required for rotor deceleration,
the Centrifuge 5920 R is also ideally
suited for laboratories processing high
sample quantities.
*This centrifuge is an in vitro diagnostic accessory and
therefore itself is an in vitro diagnostic device according
to Directive 98/79/EC of the European Parliament and the
Council dated October 27, 1998.
>
In addition to the visual inspection of
the centrifuged samples, yield and
­viability of the PBMCs obtained from
samples centrifuged in the rotor
­S-4xUniversal-Large were analyzed as
an example to confirm the quality of the
PBMC isolation using Ficoll-Paque PLUS.
The data provided by GE Healthcare,
which routinely achieved a viability of
95 % (+/−5 %) during internal testing,
served as a reference [6]. The studies
performed at Eppendorf showed an
­average viability of 94 %, which is in
line with expected values.
According to the literature, the expected
yield of mononuclear cells falls between
0.8 and 3.2 x 106 cells/mL of blood [7].
A result of 3.0 x 106 cells/mL (ramp 0/0)
or 2.16 x 106 cells/mL of buffy coat
(ramp 3/3), respectively, places the
­total yield of viable cells within a very
good range.
These results demonstrate that the
Centrifuge 5920 R fully meets the demands of low vibration performance,
independent of the swing-bucket rotor
used, and independent of whether an
acceleration/deceleration rate of 0/0 or
3/3 was selected. Furthermore, a ramp
of 3/3 allows considerable time savings
of up to 19.7 min (36 %) compared to
the centrifugation parameters recommended in the literature (deactivated
brake) (Fig. 3) [8].
[2] Bøyum A. Isolation of mononuclear cells
and granulocytes from human blood. Scan. J.
Clin. Lab. Invest. 1968, 21 (Suppl. 97): 77-89.
[3] Graham J. Biological Centrifugation: The
Basics from Background to Bench. Oxford: BIOS
Scientific Publishers Limited; 2001.
[4] Heine H, Uschkureit T. Software controlled
acceleration and braking ramp for optimized
isolation of mononuclear cells. Eppendorf
­Application Note 074;
www.eppendorf.com/applications
[5] Seeligmüller N. Faster isolation of PBMCs
using Ficoll-Paque® PLUS in the Eppendorf
Centrifuge 5920 R. Eppendorf Application Note
372; www.eppendorf.com/applications
[7] HIV/AIDS Network Coordination: CrossNetwork PBMC Processing Standard Operating
Procedure. www.hanc.info
34.6
S-4x750 (0/0)
[8] Luttman W, Bratke K, Küpper M, Myrtek D.
Der Experimentator: Immunologie. 4. Auflage.
Heidelberg: Springer Verlag; 2014.
54.3
S-4x1000 (3/3)
[1] Theml H, Diem H, Haferlach H. Taschenatlas
der Hämatologie: Morphologische und klinische
Diagnostik für die Praxis. 5. Auflage. Stuttgart:
Georg Thieme Verlag; 2002.
[6] Ficoll-Paque® PLUS Instructions.
www.gelifesciences.com
Total run times swing-bucket rotors Centrifuge 5920 R
(centrifugation time 30 min, ramps 0/0 and 3/3)
S-4x750 (3/3)
Literature
34.7
S-4x1000 (0/0)
54.1
S-4xU-Large (3/3)
33.4
S-4xU-Large (0/0)
47.36
0
10
20
30
40
50
60
Minutes
Readers’ service
Fig. 3: Total centrifugation times for density gradient centrifugation in the new Eppendorf Centrifuge 5920 R while using
different swing-bucket rotors and ramp settings, 0/0 or 3/3, respectively
Centrifuge 5920 R • Ref. no. 289
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>
(BN 45) JUNE 2016 PAGE 3
Faster and More Reproducible Cell Viability Assays
with the Eppendorf epMotion ® 96
ERIC GANCAREK, MAUD BRASSEUR, MURIEL ART, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM
HANAË A. HENKE, EPPENDORF AG, HAMBURG, GERMANY
Abstract
Cell-based assays are an established
standard tool in research. The most
common formats for these assays are
96- and 384-well plates, requiring
­multichannel pipetting to ensure quick,
efficient, and reproducible handling.
In this Application Note, a cell viability
assay was used to determine the
speed, reliability, and reproducibility
of a manual 12-channel pipette, an
electronic 12-channel pipette, and the
semi-automated 96-channel electronic
pipette epMotion 96. Compared to a
manual 12-channel pipette it was shown
that the epMotion 96 and the electronic
12-channel pipette delivered more reproducible results, with the epMotion 96
being significantly faster.
Introduction
Cell-based assays are an important
method in life science and pharmaceutical laboratories. They allow limiting the
use of animal models and providing more
representative information generated
in a biological context like a normal
physiological situation [1].
A large panel of cell responses can be
tackled by this method such as cell
proliferation, activation of specific signaling pathways, cytotoxic impact of a
molecule, or cell viability.
This could be solved by automated
­systems, but these can be an investment smaller labs cannot afford. The
epMotion 96, a semi-automated pipette
equipped with 96 channels, is an alternative solution for scientists interested
in increasing their throughput. This
study clearly illustrates the benefits of
using the epMotion 96 for cell-based
assays.
Method: cell viability assay
Cells seeded into 96-well Eppendorf
Cell Imaging Plates are grown over night
and then treated with eleven increasing
concentrations of Staurosporine (STS)
between 1 nM and 20 µM to induce cell
death. The DMSO concentration does
not exceed 1 %.
The plates are incubated for 48 h in
presence of the cytotoxic agent. After
incubation, 20 µL of freshly prepared
CellTiter-Blue® Cell Viability reagent
(Promega®) are added to each well of
the 96-well plates. The CellTiter-Blue
reagent contains highly purified resazurin. Viable cells retain the ability to
reduce resazurin into highly fluorescent resorufin. Nonviable cells rapidly
lose metabolic capacity, and thus do
not generate a fluorescent signal.
­Fluorescence is read in each well at
two wavelengths (excitation at 535 nm;
emission at 595 nm).
epMotion® 96
Results and discussion
Impact of liquid handling instrument
on cell-based assay performances
In the cell-based assay chosen for
this study, volumes dispensed were
between 10 µL and 90 µL. Moreover,
­solutions handled vary from a cell
suspension to a cytotoxic agent dissolved in DMSO. The epMotion 96
was compared to a 12-channel electronic pipette and a 12-channel manual pipette for each of the following
­experiments.
Dose-dependent viability curves
The epMotion 96, a 12-channel electronic pipette, and a 12-channel manual
pipette were used to seed HepG2 cells
into 96-well plates (three plates in
­parallel) and to add the assay reagent
to all wells. The HepG2 cells viability
was evaluated after treatment by a
STS dose-response curve.
An electronic 12-channel pipette was
used to add eight replicates of this
curve to the nine microplates. A widely
distributed technique is to remove the
data generated from curves displayed
in rows A and H in all plates, so six
­replicates were used for calculation
and are shown in Fig. 1.
Profiles generated were comparable
for each instrument used for cell seeding and reagent adding. Nevertheless,
with a manual pipetting system, the
­assay reproducibility was affected as
indicated by higher standard deviations.
Manual pipette
Electronic pipette
45,000
45,000
40,000
40,000
40,000
35,000
35,000
35,000
30,000
25,000
20,000
15,000
10,000
5,000
0
-10
Fluorescence (RFU)
45,000
Fluorescence (RFU)
Fluorescence (RFU)
>
Taken together, the epMotion 96 is the
ideal tool for cell-based assays in
plates, including cell seeding, media
change, and compound and assay
­reagent addition.
Compatibility of the liquid handling
tool with the cell-based assay has to be
demonstrated and assay reproducibility
is an essential factor which has to be
evaluated [2].
30,000
25,000
20,000
15,000
10,000
-9
-8
-7
-6
-5
log10 (Staurosporine) M
-4
25,000
20,000
15,000
10,000
5,000
5,000
0
-10
30,000
-9
-8
-7
-6
-5
log10 (Staurosporine) M
-4
0
-10
Fig. 1: Dose-dependent STS toxic effect on HepG2 cells (n=six replicates)
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
-9
-8
-7
-6
-5
log10 (Staurosporine) M
-4
>
PAGE 4 (BN 45) JUNE 2016
Faster and More Reproducible Cell Viability Assays
with the Eppendorf epMotion ® 96
Intra-plate CV
(n = 96)
Global intra-plate CV
(n = 96 x 3)
Global inter-plate CV
(n = 96 x 3)
Plate 1
Plate 2
Plate 3
epMotion® 96
9.4
7.9
8.8
8.7
9.8
Electronic pipette, 12 channels
7.2
8.4
10.1
8.6
10.4
Manual pipette, 12 channels
13.1
15.9
16.8
15.3
17.9
Table 1: Intra- and inter-plate CVs calculated for CellTiter-Blue cell viability assay for the respective instruments used (three replicates)
Intra- and inter-plates reproducibility
>
Time needed to fill one assay plate with different
liquid handling tools
20
18
epMotion® 96
Electronic pipette, 12 channels
16
Time in minutes
The epMotion 96 was used for all three
successive assay steps (cell seeding,
cell treatment by a cytotoxic agent, and
assay reagent addition). A unique STS
concentration (1,000 nM) was applied
on HepG2 cells to induce cell death.
The assay reproducibility was assessed
by calculating intra-plate and inter-plate
coefficients of variation (CV) in between
the three replicates presented in Table 1.
Manual pipette, 12 channels
14
12
10
>
The global intra-plate CVs and the interplate CVs generated by the epMotion 96
and the electronic pipette were comparable and significantly lower than CVs
obtained with a manual instrument.
These instruments allow almost complete elimination of human error as
one of the major sources of variability.
­Besides the assay reproducibility improvement, the use of an electronic
­instrument also allows to ­reduce the
hands-on time as indicated in Fig. 2.
Fig. 2: Hands-on time needed for one assay plate for each CellTiter-Blue Cell Viability assay step depending on the
instrument used
The epMotion 96 appears as the fastest
option since a complete cell-based assay
requires less than four minutes, while
with the electronic pipette one needs
approx. eight minutes and with the
manual pipette almost 20 minutes.
medium to high throughput. This instrument allows saving hands-on time
and combines the ease of use of pipettes with a high precision at a price
more affordable than fully automated
systems.
Conclusion
Literature
In this Application Note, we show the
capability to use the epMotion 96 for
processing a cell-based assay. With all
assay performance parameters studied,
it was demonstrated that the assay
­reproducibility was significantly better
when using the epMotion 96 or the
electronic ­pipette. When a large number of tests have to be handled in parallel, the epMotion 96 can represent a
good choice for scientists interested in
[1] Zang R, Li D, Tang I-C, Wang J, Yang S-T.
Cell-based Assays in High-Throughput Screening for Drug Discovery. International journal
of Biotechnology for Industries 2012; 1:31-51.
8
6
4
2
0
Cell seeding
Cytotoxic agent
addition
Assay reagent
addition
[2] An W.F, Tolliday N. Cell-based Assays for
High-Throughput Screening. Mol Biotechnol
2010; 45:180-186.
Readers’ service
epMotion® 96 • Ref. no. 275
Your local distributor: www.eppendorf.com/contact
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All assay steps
(BN 45) JUNE 2016 PAGE 5
The Tip of the Iceberg: How Pipette Tips Influence Results.
Part 2: Perfect Geometry Makes a Difference
ULRIKE GAST, EPPENDORF AG, HAMBURG, GERMANY
LAURA KOCH, EPPENDORF POLYMERE, HAMBURG, GERMANY
Abstract
We performed a study including standard tips from different
manufacturers in order to investigate the tip-related influences
on the pipetting result. The study showed a dramatic impact
of the tip on the pipetting accuracy. Small volume pipetting
accuracy is significantly influenced by the quality of the tip
orifice. Any impairment of geometry and shape can lead to
retention of liquid.
Introduction
Tip-related influencing factors on pipetting results can be
compared to an iceberg. Some influencing factors are easy
to observe, like the necessity for high tip attachment forces
in order to achieve a secure and tight fit of non-recommended
tips on the pipette cone. Other factors stay rather unknown,
like the geometry of the tip orifice.
round geometry lead to deflection of liquid drops to the outside of the thinner wall. Flashes cause liquid retention. Such
production flaws are generated within tip production by
tools with low quality or long maintenance cycles and/or a
non-optimized injection molding process.
Since the production obviously has a high impact on the geometry and shape of the tip, influencing the pipetting result,
it makes sense to take a closer look at production. The next
part of this series will focus on the production of pipette tips
and, in this context, the use of additives.
(BN 44) JANUARY 2016
PAGE 1
The Tip of the Iceberg: How Pipette Tips Influence Results.
Part 1: Tip Fit Is Not All Users Should Look for
MURIEL ART, VINCENT DUFEY, IOAN GLIGOR, EPPENDORF APPLICATION TECHNOLOGIES S.A., NAMUR, BELGIUM
ULRIKE GAST, RONJA KUBASCH, EPPENDORF AG, HAMBURG, GERMANY
Part 1 of this series of Application Notes described pipette
and tip as a system which may not perform within permissible error tolerances with all tips [1].
Material and methods
The fact that a tip fits onto a pipette
cone does not say anything about the
performance of the pipetting system
comprising the components “Pipette
and Tip”. We performed a study including standard tips from 15 different
manufacturers in order to investigate
the tip-related influence on the pipetting result. The study results showed
a dramatic influence of the tip on the
pipetting accuracy.
order to achieve efficient tip fit. Other
problems often remain unrecognized
like decreased pipette accuracy when
using other tips than recommended by
the pipette supplier.
The ISO 8655-2:2002 standard [1] defines pipette and tip as a system, which
requires extra calibration for the use of
other manufacturers’ tips. But why does
this standard put so much focus on a
product that is to be discarded after
usage?
The standard ISO 8655 regards pipette
and tip to be a system. Our study results
emphasize the validity of this statement
and the need of calibration / pipette
adjustment if other tips than recommended by pipette manufacturer are
to be used.
This series of articles answers this
question. It shows the influence of tips
on the pipetting result explaining the
main tip-related impact factors.
Introduction
Eppendorf Xplorer ® plus 50 –1,000 µL
and 0.5 –10 µL were used. Racked 10 µL
and 1,000 µL standard tips of Eppendorf
and 14 other manufacturers have been
tested. Exceptions: Manufacturer H did
not offer racked 10 µL standard tips,
manufacturers K and N offered only
1,250 µL tips for 1,000 µL pipettes.
Within the scientific community, a rising number of published experiments
cannot be reproduced by other groups.
In general, plastics are not taken seriously leading to problems with analysis
results caused by e.g. leachables or incorrect pipetting volumes. This may
lead to results not being reproducible if
performed by other groups using other
consumables.
Some problems with pipette tips are
obvious, e.g. the need to push tips
with force onto the pipette cone in
Material and methods
General material
Calibration by gravimetric method
The performance of the system “Pipette
and Tip” was determined by calibration
according to [1]. Environmental conditions were set according to requirements
[1].
Calibration was performed using analytical balance Model XP26PC (MettlerToledo®) at 100 % nominal volume and
10 % nominal volume. Two series of 10
pipettings were performed. Systematic
error and random error were determined
for each series of 10 measurements and
compared to specifications [1] and [2].
For further information please refer to
[3].
Results and discussion
While being perfectly within error limits
with Eppendorf tips, we found the system
“Pipette and Tip” to be out of specifications when using other manufacturers’
tips.
As shown in Fig. 1 and 2, the allowed systematic error was exceeded with 4 manufacturers’ tips at a volume of 1,000 µL
and with 5 manufacturers’ tips at 1 µL
test volume. These tips with 1,000 µL
test volume exceeded not only the manufacturer specifications but also the wider
limits for systematic error as stated by
the ISO 8655:2002 standard [1]. The
random error was noticeably increased
but stayed within allowed tolerances.
When comparing the calibration results
with the outcome of dimensional measurements, it becomes clear that with
1,000 µL the biggest impact factor is
the air-cushion size: Those tips that produced error limits beyond the pipette
1 µL
0.5
0.45
0.4
1.5
1
0.5
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Results and discussion
10 µL
2
Random error [%]
The orifice microscopy of 10 µL tips from Eppendorf and three
other suppliers (manufacturers E, F, and H) was performed
using a microscope (Leica®) with 25-fold magnification and
a DFC 280 digital camera (Leica).
Random error [%]
>
This is in concert with ISO 8655 [2] which declares that
­pipette and tip build a system which needs to be calibrated if
alternative tips are used. In part 1, the air-cushion size was
detected to be the main influencing factor on bigger volumes
like 1,000 µL. In contrast, with 10 µL tips other f­ actors come
into play. Here, the quality of the tip’s orifice was found to
be of high importance. For further investigation, the orifices
of tips from different manufacturers were scrutinized and
compared to calibration results [1,3].
Abstract
-4
-2
0
2
4
6
8
10
0
-1.5
Systematic error [%]
-1
-0.5
0
0.5
1
1.5
Systematic error [%]
Fig. 1: Calibration results using 10 µL tips of different manufacturers. The red shaded area shows the span of the maximum permissible errors stated for the system “Pipette and Tip”
by pipette supplier. All data points within the red shaded area are within the specifications.
The zone where liquid leaves the tip during dispensing is
very important for the accuracy of results. At this part of the
tip, the drop cut-off occurs. Any imperfection of geometry or
shape, e.g. by production errors, can lead to liquid retention.
This especially plays a role with small volumes. A poor drop
cut-off may not only impair the pipetting result but can make
it impossible to dispense small volumes below 1 µL onto a
solid surface.
Fig. 1 (see next page) shows examples of such production
flaws. Tips from these manufacturers lead to the system
­performing outside permissible systematic error tolerances
[1,3]. This underlines the direct influence of tip orifice geometry on pipetting of small volumes.
A good tip has a perfectly round geometry and uniform wall
thickness. The orifice does not show flashes or loose material
residues. Walls with varying thickness and not perfectly
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Missed part 1?
A rising number of published experiments cannot be reproduced
by other groups. In general, only little attention is paid to the
purchase of plastic laboratory consumables, like pipette tips and
microtest tubes.
The fact that a pipette tip fits onto a pipette cone does not say
anything about the pipetting accuracy of the pipetting system
comprising the components “Pipette and Tip”. We have studied
this issue thoroughly in part 1 of our series “The Tip of the Iceberg:
How Pipette Tips Influence Results”.
If you missed part 1, then check out BioNews No. 44 in our
­archive at www.eppendorf.com/bionews.
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
>
PAGE 6 (BN 45) JUNE 2016
The Tip of the Iceberg: How Pipette Tips Influence Results.
Part 2: Perfect Geometry Makes a Difference
Lying flash
Flash
Eppendorf epT.I.P.S.® 10 µL
Manufacturer E
The orifice has a good geometry and the function is not negatively
influenced by production errors.
Problem 1: Lying flashes caused by non-harmonized ejection molding
process. Cavity has not been fully filled with liquid PP.
Result: Risk of deflection of water drop because of varying diameter
of frontal area
Problem 2: Flashes at exterior wall
Result: Risk of keeping liquid residues
Molding flash
>
Flash
Molding flash
Manufacturer F
Manufacturer H
Problem 1: Flashes at interior and exterior wall caused by long
maintenance cycles of tool.
Problem 1: Molding flashes caused by a non-tightening tool or problems with injection molding process where too much liquid PP has
been injected.
Result: Risk of liquid residues and risk of PP particles falling into
sample. Additionally, particles inside the tip displace water leading
to wrong liquid volume. Such an error pattern makes it impossible
to dispense e.g. 0.8 µL sample onto a solid surface.
Result: Liquid retention
Problem 2: Noticeable core shift. Not all walls have the same thickness. This error pattern can be caused by e.g. a poorly manufactured
tool.
Result: Risk of deflection of liquid beside instead of into the target
vessel.
Fig. 1: Microscopic pictures of 10 µL tip orifices of different manufacturers. Manufacturer E, F, and H failed the calibration by impaired systematic error. The examples have been
chosen to explain production errors. PP = polypropylene.
Literature
[1] The Tip of the Iceberg: How Pipette Tips Influence Results. Part 1:
Tip Fit Is Not All Users Should Look for. BioNews 44, January 2016.
www.eppendorf.com/bionews
[2] DIN EN ISO 8655:2002. Piston-operated volumetric apparatus.
Beuth-Verlag, Berlin, Germany
[3] Application Note 354: The Tip of the Iceberg: How Pipette Tips
­Influence Results. www.eppendorf.com/applications
Readers’ service
Eppendorf Liquid Handling Consumables • Ref. no. 288
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
>
(BN 45) JUNE 2016 PAGE 7
Improved Automated Single Cell Clone Analysis with the
SynenTec Cellavista® System and Eppendorf Cell Culture Plates
MAREIKE PANZ, JOHANNA PRIEB, SYNENTEC GMBH, ELMSHORN, GERMANY
INES HARTMANN, EPPENDORF AG, HAMBURG, GERMANY
Abstract
Single cell cloning is one critical step in
stable cell line development. To ensure
the monoclonal character of the population, a reliable microscopic identification
of single cells is essential. Automated cell
analyzers like the SynenTec Cellavista
System (Fig. 1) allow a high throughput
and a reliable tracking and documentation from single cell status to colony
formation.
the method it is critical to verify that the
resulting colony derives from a single
cell. Manual microscopic observation of
single cell clones and colony formation
can be time-consuming, unreliable, and
difficult to document. Automated cell analyzers like the SynenTec Cellavista System can monitor cell growth of colonies
from single cell status by repeated measurements of the same microplate over
a span of typically two to three weeks.
The system is able to follow the growth
of the clones and can completely document this process. Here we analyze the
influence of the consumable in single cell
cloning analysis. A uniform well illumination without interfering shadows at the
well edges is important to detect cell
colonies growing at the well periphery.
The choice of the consumable can affect the analysis as well: Evaporation
and meniscus effects can lead to optical interferences with the result that
single cell clones may stay undetected
or be falsely analyzed. In Eppendorf
Cell Culture Plates (Fig. 2), the liquid
meniscus is minimized resulting in an
excellent well illumination.
Introduction
Stable transfected cell lines arisen from
one single cell clone allow continuous
gene expression by a genetically homogeneous population. Independent from
Fig. 2: The Eppendorf 96-Well Cell Culture Plate is equipped
with an outer moat that can be filled with liquid to insulate
specifically the edge wells to minimize evaporation.
The inter-well space and the outer moat
of the Eppendorf Cell Culture Plates
were filled with sterile, distilled water
to minimize evaporation of medium over
time. Plates were kept in the incubator
until cell seeding. Competitor plates
were placed in the incubator at the
same time prior to seeding. Adherent
CHO-GH3 cells were prepared and
seeded in a concentration of 0.5 cells
per well in 200 μL medium.
Day 0
Images (n=4) were taken on day 0 and
day 1 followed by measurements every
2 – 3 days until day 10 –14 post-seeding.
In order to prevent cell loss no change
of medium was performed.
Meniscus effect
Plates were seeded with different
­volumes of medium from 1–6 μL/mm2.
Volumes were adapted to volume/surface area to cover variances in growth
areas of plates. In a second ­experiment
cells were seeded using 3.31 μL/mm2
and stained with trypan blue. All plates
were analyzed using the Cellavista
­System.
Edge effect
Single cell seeding procedure
Fig. 1: The automated cell analyzer SynenTec Cellavista
System
Edge effect
Results and discussion
Materials and methods
>
Analysis
Day 1
Evaporation in 96-well plates is a critical
factor. With the Eppendorf Cell Culture
Plates evaporation can be reduced to a
minimum [1].
Fig. 3 shows that filling the moat and
the inter-well space of the Eppendorf
plate leads to clear well illumination
throughout the entire duration of incubation. Colony growth in the edge wells
of the plate can be detected clearly
throughout the whole incubation time.
Day 6
Day 14
Fig. 3: Well brightness and illumination uniformity. Outer well 6A of the Eppendorf 96-Well Cell Culture Plate was measured at indicated time points using the Cellavista System. Filling of the outer moat minimizes evaporation and enables
optimal illumination of the well also after 14 days of incubation. The complete colony is clearly visible even in the well
edge. (Upper panel: 4x objective, brightfield; lower panel: image detail)
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
>
PAGE 8 (BN 45) JUNE 2016
Improved Automated Single Cell Clone Analysis with the
SynenTec Cellavista® System and Eppendorf Cell Culture Plates
Competitor
Eppendorf
1 µL/mm2, BF 1
2 µL/mm2, BF 1
3 µL/mm2, BF 1
4 µL/mm2, BF 2
5 µL/mm2, BF 3
6 µL/mm2, BF 3
Fig. 4: Comparison of meniscus effect in Eppendorf Cell Culture Plates (96-Well, TC-treated) and competitor plates. The lower the volume the more critical is the dark shadow formation.
Volumes were adapted to volume/surface area (Cellavista, 2x objective, brightfield adjustment: BF1 = 8 ms, BF2 =12 ms, BF3 = 20 ms).
Competitor
Eppendorf
>
No shadow visible
Shadow visible
Fig. 5: Comparison of meniscus effect with trypan blue stained cells. No shadow at the edge is visible in the Eppendorf Cell Culture Plate. Volumes were adapted to volume/surface area
(Cellavista, 2x objective, brightfield).
Meniscus effect
Conclusion
Literature
A common problem when imaging 96well formats is the meniscus formation
of the liquid. The curvature of the liquid
causes a refraction of the light with the
result of uneven illumination of the
sample which may lead to a distorted
image as if looking through a porthole.
To analyze the meniscus effect, different
liquid filling heights were measured in
the tested plates.
Single cell cloning using SynenTec’s
automated Cellavista analyzing system
together with the Eppendorf Cell Culture
Plates is best suited for successful single
cell clone analysis. The Cellavista can
monitor cell growth of colonies from
single cell status by repeated measurement of the same microplate over a
­defined span. By this, documentation
of colony development is made easy.
Eppendorf Cell Culture Plates have an
optimized microscopic performance:
The reduced meniscus enables a uniform
illumination without disturbing shadows
at the edges. Due to the possibility to
reduce evaporation in the plate to a minimum, Eppendorf Cell Culture Plates
enable an improved microscopic performance also in long-term incubation
experiments.
[1] Wagener J, Plennevaux C. Eppendorf 96Well Cell Culture Plate – A simple method of
minimizing the edge effect in cell-based assays.
Eppendorf Application Note 326;
www.eppendorf.com/applications
Fig. 4 shows that the Eppendorf plate
allows an excellent illumination of the
whole well area also at reduced filling
heights. In comparison to that the competitor plate already shows disturbing
shadows and uneven illumination when
using higher filling levels.
In Fig. 5 trypan blue stained cells were
analyzed in Eppendorf plates and comparable plates from a competitor using
the Cellavista Imaging System. An interfering shadow can be observed in the
competitor plate at the edge of the well
whereas in the Eppendorf plate the
shadow is reduced to a minimum.
Readers’ service
Eppendorf Cell Culture Consumables • Ref. no. 270
Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
>
STATE-OF-THE-ART DNA MEASUREMENT: EFFICIENT, FAST, AND REPRODUCIBLE · STRAIGHT FROM THE LAB
9
SOPHIE MANUELLO, EPPENDORF AG
State-of-the-Art DNA Measurement:
Efficient, Fast, and Reproducible
Molecular biologists consider quantification of dsDNA an essential routine technique. Measurement data obtained
are of great importance for subsequent experiments. In a worst case scenario, faulty underlying data may lead
to the failure of downstream applications. The Eppendorf BioSpectrometer® fluorescence combines UV/Vis and
fluorescence measurements in a single device to reliably determine both concentration and purity of your DNA
samples.
>
Why not quantify using UV/Vis
­spectroscopy?
What does fluorescence
have to do with it?
Is fluorescence the only method
­required for quantification?
Increasing discussion within scientific
forums points to the fact that UV/Vis
spectroscopy overestimates dsDNA
concentrations and that especially small
amounts are not quantified correctly.
The reason is that all nucleic acids absorb
light at 260 nm; therefore no general
distinctions can be made between dsDNA,
ssDNA, and RNA.
Certain fluorophores can only fluoresce
if bound to a specific molecule. Fluorescence kits take advantage of the binding
specificity to dsDNA, ssDNA, or RNA
and thus create a direct correlation between signal and concentration. Owing
to the high binding affinity to the target
molecule, quantification via fluorescence
is also considerably more sensitive.
In addition to the exact concentration of
dsDNA, certain downstream applications
require a statement on sample purity.
Fluorescence measurements do not provide this information. Sample purity may
be determined by UV/Vis spectroscopy
using the ratios A260/A280 and A260/
A230 as well as the shape of the spectrum.
The combination makes the difference!
UV/Vis spectroscopy and fluorescence
are methods which complement each
other. The Eppendorf BioSpectrometer
fluorescence is the first instrument on the
market to combine both methods of measurement in one small, compact device.
In combination with the microvolume cuvette Eppendorf µCuvette® G 1.0, the field
of application for dsDNA measurement is
now completed for a range from 1 pg/µL
to 1,500 ng/µL.
Several videos on the topic of “Detection”
are available at www.eppendorf.com/
playlist-detection or by QR-Code.
Eppendorf BioSpectrometer ® • Ref. no. 242
>
10 STRAIGHT FROM THE LAB · ROBUST AND RELIABLE: EASYPET® 3
HANAË A. HENKE, EPPENDORF AG
Robust and Reliable:
­Easypet® 3
Tip
Supporting You
Maintenance and Certification Services
Preventive Maintenance is a schedule of
planned service measures aimed at the
When conducting sterile work in cell culture, microbiology, or diagnos-
prevention of unexpected instrument
tics, the use of cleaning and disinfection agents cannot be avoided. For
downtimes and failures through routine
this reason, we have tested our electronic pipette controller Easypet 3 for
problems.
­resistance to 67 of the most common laboratory chemicals.
Certification Services include calibration,
maintenance and early detection of
verification, and Installation & Operational
Qualification (IQ/OQ) to ensure that your
results are consistent and reliable.
Furthermore we offer support to ensure
For clear distinction, materials were
classified as either “resistant”, showing
“limited resistance” or “increased risk/
wear”.
>
compliance with your laboratory guidelines
by providing the necessary certification
documents.
>
The test results
In our test, all parts of the Easypet 3
which could come into direct contact
with one of the test chemicals proved to
be “resistant” to almost all chemicals
tested. These test results offer you the
safety you need when working with
chemicals; for example when you use
your Easypet 3 in a cell culture or molecular biology laboratory, as well as in the
chemical or biotechnology industries.
Test report for downloading
In addition to commercial cleaning agents
and disinfectants, this included saline
solutions, buffers, acids and bases as
well as organic solvents.
The detailed report “Chemical Resistance
Easypet® 3” may be downloaded as a PDF
file at www.eppendorf.com/easypet
­under “Downloads/manuals”.
When performed on a regular basis, our
Performance Plans* are designed to keep
your Liquid Handling, Sample Handling,
and Cell Handling instruments in best
condition over years and to meet manufacturer accuracy and precision specifications:
>> ESSENTIAL CHECK
>> ADVANCED MAINTENANCE
>> PREMIUM SERVICE
>> Installation Qualification (IQ)
>> Operational Qualification (OQ)
For more information and service requests
go to www.eppendorf.com/epServices.
Rigorous testing
All individual parts of the Easypet 3
were tested separately. This approach
provided certainty that all parts displayed
comparable resistance to the chemicals
tested. Individual parts are made from
very different materials such as silicone
(pipette adapter), polypropylene (aspiration and dispensing buttons, housing,
wall holder, and shelf stand), or cyclic
olefin copolymer (battery status display).
Eppendorf Performance Plans
Easypet ® 3 • Ref. no. 250
*Performance Plans are available in selected
­countries and services may vary.
EPPENDORF TUBES® 5.0 ML – NOW ALSO AVAILABLE WITH SCREW CAP · INNOVATION 11
NILS GERKE, EPPENDORF AG
Eppendorf Tubes® 5.0 mL –
Now Also Available with Screw Cap
With the development of the first Eppendorf Tubes over 50 years ago, Eppendorf ignited the evolution of the
­reaction tubes. Since that time, Eppendorf Tubes have been used worldwide on a daily basis for routine and
­special procedures. In order to further optimize applications in the life sciences, the Eppendorf Tubes 5.0 mL
were developed, thus closing the gap between existing tube size variants. When working in the medium volume
range, with this new tube at hand, it is no longer necessary to handle large tube formats which can often be
­impractical, inconvenient, and prone to contamination.
With system
Naturally, all these advantages can only be enjoyed if seamless
integration into the existing laboratory environment, with all its
established laboratory protocols, is possible. This is achieved
by the carefully thought-out Eppendorf 5.0 mL system. The
comprehensive solution with matching accessories allow all
common laboratory workflows to be carried out in an easy,
practical, and cost-efficient manner.
>
Eppendorf offers custom system components for efficient and
easy pipetting, gentle mixing, fast centrifugation, incubation,
automated liquid handling as well as secure storage. Furthermore, thanks to its conical shape, the Eppendorf Tube 5.0 mL
is compatible with the accessories for the common 15 mL
tubes, i.e. many adapters and racks can be utilized.
Eppendorf Tubes 5.0 mL: with snap cap or screw cap for individual application demands in
the medium volume range
With screw cap
The introduction of the screw cap tube represents the next
major innovation within the Eppendorf Tubes 5.0 mL system.
In addition to utmost seal security, the grooved, multi-surface
sides of the newly designed screw cap ensure a slip-free grip.
These tubes complete the application spectrum for sample
processing in the medium volume range. They are especially
suitable for applications where a screw cap can be beneficial
to either experiment or handling:
>> Long-term storage and archiving of samples and solutions at
­temperatures as low as −86 °C
>> Secure sample transport
>> Incubation at high temperatures up to 100 °C
In addition they are well suited for sample preparation requiring
centrifugation – the new Eppendorf Tubes 5.0 mL with screw cap
feature the same centrifugation stability of up to 25,000 x g as
the 5.0 mL snap cap tubes. Now the user can choose the ideal
tube for each individual application and thus further optimize
laboratory protocols.
The 5.0 mL system is perfectly adapted to the Eppendorf Tubes 5.0 mL
More information at www.eppendorf.com/5mL
Eppendorf Tubes® 5.0 mL • Ref. no. 264
>
12 STRAIGHT FROM THE LAB · HOLISTIC APPROACH TO ERGONOMICS
TANJA MUSIOL AND JAN-HENDRIK BEBERMEIER, EPPENDORF AG
Holistic Approach to
­Ergonomics
Tip
Eppendorf Services
Our online Service & Support portal
www.eppendorf.com/epServices is not
only THE gate to Eppendorf’s comprehen-
Since the early 1970s, further to considering the technical specifications
of its products, Eppendorf has also incorporated their ergonomic properties
within their respective work environments. In 2003, the holistically de-
sive service offerings. It also features a
great deal of additional useful information.
Application Support
Our highly trained Scientific Support Staff
signed PhysioCare Concept® for Liquid Handling products was launched
is available to assist you with any questions
and brought with it a fresh perspective on daily laboratory processes.
concerning your Eppendorf products.
Seminars and Training
Find the latest information on seminars
and webinars conducted by experienced
specialists. Our courses have a practical
orientation and will help you master your
daily lab routine challenges.
Technical Support
>
For maintenance, certification and repair
service, please contact your local service
organization here.
Maintenance and Certification Services
Learn more about the Eppendorf Performance Plans. The information is sorted
by product groups and structured in a
clear and concise manner. For conveniently
Since that time, the PhysioCare Concept
has seen continuous development as well
as expansion to other product groups.
Today it links the three spheres of action
in the lab: the user, the laboratory, and the
laboratory workflow, with the goal of harmonizing the workflow in the laboratory
with the health and well-being of the staff.
Ergonomics encompasses more aspects
than meets the eye. For example, with
manual Eppendorf pipettes, probably the
most well-known products with PhysioCare Concept features, the focus is on the
pipetting force: The smaller the force
needed for pipetting, the less strain will
be imposed on the muscles, tendons, and
joints. In Eppendorf laboratory instruments, on the other hand, features like
easy-to-read displays, simple operation,
and intuitive menu navigation as well as
low noise levels contribute to the overall
ergonomics.
One further crucial component of ergonomics is laboratory organization: How
do I prevent repetitive stressful movements, unnecessary walking, or frequent
interruptions of my work? How do I optimize the organization of my work space?
Eppendorf, with its decades of experience, will offer useful ideas that are easily
put into practice.
Conclusion
The PhysioCare Concept offers holistic
solutions for optimizing the workflow
in the laboratory. Ergonomic product
features are complemented by recommendations on how to equip and organize
ergonomic workflows. The result: an improved work environment, paired with
higher efficiency.
More information at
www.eppendorf.com/physiocare
getting in touch with the Eppendorf Service
Team, you’ll find easy to use contact forms.
Quality and Regulatory Affairs
Your source for certificates, material
safety data sheets, and information on
purity grades of Eppendorf consumables.
Knowledge Base
The Knowledge Base is a true treasure box!
Download all kinds of literature, such as
operating manuals, application notes,
protocols, posters, user guides, white
papers, just to name a few. Register or
update Eppendorf software. Check out our
FAQ database. Visit the Media Center to
watch the latest videos from Eppendorf, or
download our mobile apps.
>
EPPENDORF STERILIZE-IN-PLACE (SIP)BIOPROCESS SYSTEMS · STRAIGHT FROM THE LAB 13
SOPHEAP SUN, EPPENDORF, INC., ENFIELD, USA
ULRIKE BECKEN, EPPENDORF AG, BIOPROCESS CENTER EUROPE, JUELICH, GERMANY
Eppendorf Sterilize-in-Place (SIP)
Bioprocess Systems
Small footprint
Critical to the success of industrial bioproduction, the scale-up of bioprocesses
is currently receiving much attention.
Eppendorf offers comprehensive fermentor and bioreactor portfolios for pilot
through production applications.
Lab space is precious. That is why
­Eppendorf SIP systems were constructed
with a small footprint. At just 116 cm
wide x 86 cm deep (45.5 x 34.0 in), the
compact BioFlo 510 can fit on a lab
bench. Also the other SIP systems are
designed to make efficient use of space.
Bioprocessing is indispensable for manifold industrial applications, like the production of recombinant therapeutic proteins in mammalian cells, the conversion
of biomass into biofuels by bacteria or
fungi, vaccine production, the fermentative production of building blocks for
­bioplastics, and various others.
>
Flexible design
Often process requirements change over
time. Eppendorf SIP systems provide
much needed flexibility. A large number
of ports in the vessel head plate and
sidewall allow adjustments. The systems
facilitate bioprocessing in batch, fedbatch, and continuous mode.
The optimization of strains and cell
lines, culture conditions, and process
parameters is usually carried out in small
volumes. But once a process is established, process development scientists
will scale up for production. With its
comprehensive portfolio of sterilize-inplace (SIP) fermentors and bioreactors,
Eppendorf bridges the gap between process development in the milliliter scale
and pilot/production manufacturing.
The customer can choose from various
impeller options optimized, for example,
for fermentation, the cultivation of shearsensitive cells, or cell culture on microcarriers. Many of the options can also be
field-upgraded if the customer needs to
make a configuration change.
Fig. 1: The Eppendorf New Brunswick BioFlo Pro
A variety of vessels altogether covers a
volume range of 5.5 L to 2,400 L (Table 1).
Results gained by the Eppendorf bio­
process application team as well as data
gathered by our customers demonstrate
the excellent scale-up capabilities of
these systems.
Our SIP systems portfolio comprises the
New Brunswick™ BioFlo® 510, 610, and
Pro fermentors for microbial applications
and the New Brunswick CelliGen® 510
and Pro bioreactors for cell culture
(Fig. 1, Table 1).
BioFlo 510
BioFlo 610
BioFlo Pro
CelliGen 510
CelliGen Pro
5.5 – 32 L
13 – 100 L
32 – 2,400 L
5.5 – 32 L
18.8 – 520 L
Bacteria/yeasts/fungi
•
•
•
Plant cells/algae
•
•
•
•
•
Mammalian cells
•
•
Stem cells
•
•
•
•
Working volume
Insect cells
•
Table 1: Eppendorf SIP bioprocess systems
•
•
Reliable function
Industrial processes demand reliable
fermentor and bioreactor systems.
­Eppendorf strives to fulfill this expectation by delivering robust devices to
­minimize maintenance and downtime
and by applying rigorous quality control.
Eppendorf service begins with short
­delivery times and comprises training
and ongoing support to enable the user
to make the most of their equipment.
Brochure “We Know Bioprocessing” • Ref. no. 274
>
14 NEWS · EPPENDORF PRIZE WINNERS 2015/2016 SHIGEKI WATANABE & ADRIAN LISTON
CAROLYN TAUBERT AND BERRIT HOFF, EPPENDORF AG
Eppendorf Prize Winners 2015/2016
Shigeki Watanabe & Adrian Liston
>
Congratulations to the new winners of the Eppendorf research
prizes! The Japanese scientist Shigeki Watanabe, Ph.D., (Johns
Hopkins University, Baltimore, USA) won the 2015 Eppendorf
& Science Prize for Neurobiology endowed with 25,000 US$ for
his work on vesicle endocytosis. Shigeki Watanabe has developed two novel techniques in electron microscopy that allow
visualization of protein and membrane dynamics at synapses
at a millisecond temporal resolution. Using these techniques, he
has discovered an ultrafast mechanism that removes synaptic
vesicle components rapidly from the plasma membrane following
exocytosis and further demonstrated that synaptic vesicles are
regenerated from endosomes.
Shigeki Watanabe has started his own lab where his research
will focus on understanding the mechanisms underlying the
rapid reorganization of membrane, proteins, and organelles
that mediate synaptic plasticity.
More information at www.eppendorf.com/prize
The 2016 Eppendorf Award for Young European Investigators
went to Prof. Adrian Liston, Group leader at VIB Translational
Immunology Lab, University of Leuven, Belgium, for his seminal
work in elucidating key mechanisms by which the immune system avoids attacking its own organism while remaining effective
against pathogens. His experiments have paved the way for
understanding key steps in controlling regulatory T-cells that
are critical for balancing between autoimmunity and immunosuppression. His work opens up the way for new therapeutic
approaches towards diseases resulting from a dysregulated
immune homeostasis.
In the Translational Immunology Lab, Adrian Liston and his
team work on both the discovery (Treg biology, diabetes) and
applied (genetics, human disease) aspects of translational
­immunology.
More information at www.eppendorf.com/award
Both prize winners will visit Eppendorf in Hamburg during 2016.
Check out the next BioNews issue for more info!
Trademark Information
Amazon® is a registered trademark of Amazon Tech, Inc., USA. CellTiter-Blue ® and Promega® are registered trademarks of Promega Corporation, USA. Cellavista® is a registered
trademark of SynenTec GmbH, Germany. Ficoll-Paque ® is a registered trademark of GE Healthcare Bio-Sciences AB, Sweden. GE Healthcare ® is a registered trademark of General
Electric Company, USA. Leica® is a registered trademark of Leica Microsystems IR GmbH, Germany. Nalgene ® is a registered trademark of Thermo Fisher Scientific, USA. Triton™
and Tween™ are trademarks of Thermo Fisher Scientific Inc., USA.
Eppendorf ®, the Eppendorf logo, Combitips®, Easypet ®, ep Dualfilter T.I.P.S.®, epMotion®, Eppendorf µCuvette®, Eppendorf BioSpectrometer ®, Eppendorf Reference ®, Eppendorf Tubes®,
epPoints ®, the epServices ® logo, epT.I.P.S.®, FastTemp pro ®, Multipette ®, PhysioCare Concept ®, and Repeater ® are registered trademarks of Eppendorf AG, Germany. ConvexAccess™,
Eppendorf Quality™, and New Brunswick™ are trademarks of Eppendorf AG, Germany. BioFlo ® and CelliGen® are registered trademarks of Eppendorf, Inc., USA.
>
PRIZE COMPETITION · SERVICE 15
Win a Multichannel Pipette!
The solution of the prize competition of BioNews No. 43 was
“Cell Handling Quality Products“. Damjan Franjevic (University of Zagreb, Croatia) won the first prize.
Good luck in our new competition!
How to find out the solution: Simply arrange all letters in the
light gray boxes of the crossword in the correct order. Send us
the solution until October 31, 2016.
1
2
3
4
5
6
10
14
22
30
>
1st Prize:
1 Eppendorf Reference® 2
Pipette (multichannel,
adjustable-volume) of your
choice
19
24
26
27
2nd to 5th Prize:
29
31
32
35
36
39
18
23
25
28
9
12
17
21
8
15
16
20
All correct answers will be considered for a prize. Winners will
be notified in writing. Cash payment of the prize is not possible. No recourse to legal action. The judges’ decision is final.
­Eppendorf employees and their families may not participate.
The winner of the first prize will be published in BioNews No. 47.
7
11
13
You can either send us an e-mail to [email protected],
or participate online at www.eppendorf.com/bn-service.
33
1 Amazon® Voucher
worth 50.00 EUR
34
37
38
40
41
42
43
44
6th to 15th Prize:
45
46
47
48
51
52
53
49
400 bonus epPoints® each
50
(epPoints registration required)
ACROSS
DOWN
1 Opportunity, possibility
6 President of the United States
2009 –2016
10 Website accepting changes,
contributions, corrections
12 Short form of Samuel
13 Chinese politician
14 Devoted to technical subjects,
computers, etc.
16 What do rubidium and rhythm and
blues have in common
17 Software optimizer
20 System of signals/symbols for
communication
22 Transcription factor (abbrev.)
24 17th letter of the Greek alphabet
25 Used in molecular biology (when
magnetic)
27 Group of people working together
29 Alcoholic drink
30 German capital
32 Amyotrophic lateral sclerosis
(abbrev.)
35 Immunoglobulin (abbrev.)
36 Electronic pipette controller
39 Gemstone
41 ISO country code for Turkey
42 Area of land for recreation
45 French personal pronoun
46 Postscript (abbrev.)
47 Source of trouble or worry
50 ISO country code for Spain
51 Saint (abbrev.)
52 Standard deviation (abbrev.)
53 A royal hall in London is named after
him
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25
26
27
28
Curriculum vitae (abbrev.)
Prize, medal
Tantalus' daughter
Creatine kinase (abbrev.)
Developed the general theory of
relativity
Chemical symbol for arsenic
Finely ground green tea
Before noon (abbrev.)
Chemical symbol for strontium
Dots per inch (abbrev.)
Italian for three
One's domicile, house
Positive-displacement tips
State in the Northwest region of the
United States (abbrev.)
Frequently asked questions (abbrev.)
Severe snowstorm
French writer in the 19th century
Flat piece used for carrying things
Chemical symbol for argon
31 Egomaniac, ego-tripper (syn.)
33 Treasure made of vinyl, reaches
33 1/3 rpm
34 Type of gundog
37 Hard metal
38 Confidence, faith
40 Computer program running on
mobile devices (pl.)
43 ISO country code for Romania
44 1,000 base pairs
48 Home of CSULA, CSUN, USC,
UCLA, etc. (abbrev.)
49 1,000 times 44 down
Solution hint for prize competition of BioNews No. 45:
E
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A
I
Send us the solution until October 31, 2016, via e-mail to
[email protected], or participate online at www.eppendorf.com/bn-service.
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