No. 43 – 2015 (BN 43) JULY 2015 PAGE 1 A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels Abstract In recent years single-use bioreactors gained more and more importance in animal and human cell culture. With the new line of BioBLU f rigid wall, stirred- tank single-use vessels Eppendorf of- fers premium solutions for microbial applications. In the following case study, reproducible process control was achieved with par- allel operated BioBLU 0.3f single-use and reusable glass vessels, both used in an Eppendorf DASboxMini Bioreactor System (Fig. 1). Fermentation of E. coli K12 led to very comparable results thus proving the tested single-use vessels to be an ap- propriate tool to accelerate microbial process development and shorten time- to-market in all industries related to microbial production processes. Introduction Single-use bioreactors are a suitable tool for time and cost effective biopro- cessing. Minimized setup times, elimi- nated cleaning procedures and there- fore reduced labor time can sustainably accelerate bioprocess development. In all biopharmaceutical industries single- use technologies are widely used in mammalian cell culture. With the new BioBLU f line, specifically designed to meet the needs of fermen- tation, single-use bioreactors make their way to microbial applications in bio- pharma, food, and cosmetics industries. Microbial applications make specific demands on the bioreactor design and functionality. Fermentation processes need much higher k a values for proper mass transfer and suitable heating and cooling options as well. This comparative study investigates the functionality and reliability of a BioBLU 0.3f single-use mini bioreactor and an autoclavable DASbox Mini Bio- reactor (Fig. 2) in a small scale E. coli fermentation. Materials and methods E. coli K12 (DSM 498) was cultivated in a fully instrumented Eppendorf BioBLU 0.3f single-use mini bioreactor and compared to fermentations in con- ventional autoclavable glass bioreactors. The ready-to-use rigid wall stirred-tank single-use bioreactors, specifically designed for microbial applications, are equipped with a 2x Rushton-type impeller, liquid-free Peltier exhaust condensation and direct drive for high performance agitation. The overhead- driven autoclavable DASbox Mini Bio- reactors included 2x Rushton-type impeller and liquid free Peltier exhaust condensers as well. A 4-fold parallel DASbox Mini Bioreactor System with active heating and cooling capacities was used with DASGIPControl Software (now DASwarecontrol 5) for precise process control. Starting with a working volume of 100 mL each, the cultures were grown for 40 h in PAN medium with an initial glucose con- centration of 40 g/L and fed with 50 % glucose solution in the fed batch phase. The temperature was controlled at 37 °C and pH was adjusted to 6.8 via 4 % ammonia solution; the cultures were submerged aerated with a constant rate of 1 vvm (6 sL/h or 0.1 sL/min). The dissolved oxygen was maintained at 30 % with the stirrer speeds ranging from 600 rpm to 2,000 rpm which equals to tip speeds of 0.94 m/s to 3.14 m/s. Exhaust concentrations were measured and corresponding oxygen transfer rates (OTR) were automatically calculated using a DASGIP GA4 exhaust analysis module. Results and discussion Highly reproducible OTR values of up to 250 mmol/L/h were observed in the single-use as well as in the glass biore- actors (Fig. 3). CLAUDIA M. HUETHER-FRANKEN* AND SEBASTIAN KLEEBANK, EPPENDORF AG, BIOPROCESS CENTER, JÜLICH, GERMANY *CORRESPONDING AUTHOR: [email protected]BioFlo ® 320: Smart Solution for Bioprocess Control > Workflow Solutions for Molecular Biology > Eppendorf HeatSealer: Effective protection against Evaporation > Further Education with Eppendorf Application Notes Protein Sample Preparation in Eppendorf Tubes ® 5.0 mL · Microinjection into Plant Cells of Etiolated Seedlings Using the Eppendorf InjectMan ® 4 · etc.
Transcript
No. 43 – 2015
(BN 43) JULY 2015 PAGE 1
Your local distributor: www.eppendorf.com/contact Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels
Abstract
In recent years single-use bioreactors gained more and more importance in animal and human cell culture. With the new line of BioBLU f rigid wall, stirred-tank single-use vessels Eppendorf of-fers premium solutions for microbial applications.
In the following case study, reproducible process control was achieved with par-allel operated BioBLU 0.3f single-use and reusable glass vessels, both used in an Eppendorf DASbox® Mini Bioreactor System (Fig. 1).
Fermentation of E. coli K12 led to very comparable results thus proving the tested single-use vessels to be an ap-propriate tool to accelerate microbial process development and shorten time-to-market in all industries related to microbial production processes.
Introduction
Single-use bioreactors are a suitable tool for time and cost effective biopro-cessing. Minimized setup times, elimi-nated cleaning procedures and there-fore reduced labor time can sustainably accelerate bioprocess development. In all biopharmaceutical industries single-use technologies are widely used in mammalian cell culture.
With the new BioBLU f line, specifically designed to meet the needs of fermen-tation, single-use bioreactors make their way to microbial applications in bio-pharma, food, and cosmetics industries.
Microbial applications make specific demands on the bioreactor design and functionality. Fermentation processes need much higher kLa values for proper mass transfer and suitable heating and cooling options as well.
This comparative study investigates the functionality and reliability of a BioBLU 0.3f single-use mini bioreactor and an autoclavable DASbox Mini Bio-reactor (Fig. 2) in a small scale E. coli fermentation.
Materials and methods
E. coli K12 (DSM 498) was cultivated in a fully instrumented Eppendorf BioBLU 0.3f single-use mini bioreactor and compared to fermentations in con-ventional autoclavable glass bioreactors.
The ready-to-use rigid wall stirred-tank single-use bioreactors, specifically designed for microbial applications, are equipped with a 2x Rushton-type impeller, liquid-free Peltier exhaust condensation and direct drive for high performance agitation. The overhead-driven autoclavable DASbox Mini Bio-
reactors included 2x Rushton-type impeller and liquid free Peltier exhaust condensers as well. A 4-fold parallel DASbox Mini Bioreactor System with active heating and cooling capacities was used with DASGIP® Control Software (now DASware® control 5) for precise process control.
Starting with a working volume of 100 mL each, the cultures were grown for 40 h in PAN medium with an initial glucose con-centration of 40 g/L and fed with 50 % glucose solution in the fed batch phase. The temperature was controlled at 37 °C and pH was adjusted to 6.8 via 4 % ammonia solution; the cultures were submerged aerated with a constant rate of 1 vvm (6 sL/h or 0.1 sL/min). The dissolved oxygen was maintained at 30 % with the stirrer speeds ranging from 600 rpm to 2,000 rpm which equals to tip speeds of 0.94 m/s to 3.14 m/s. Exhaust concentrations were measured and corresponding oxygen transfer rates (OTR) were automatically calculated using a DASGIP GA4 exhaust analysis module.
Results and discussion
Highly reproducible OTR values of up to 250 mmol/L/h were observed in the single-use as well as in the glass biore-actors (Fig. 3).
CLAUDIA M. HUETHER-FRANKEN* AND SEBASTIAN KLEEBANK,
Fig. 1: DASbox Mini Bioreactor System for microbial applications equipped with BioBLU 0.3f Single-Use Vessels and autoclavable DASbox Mini Bioreactors with Rushton-type impeller. Fig. 2: BioBLU 0.3f Single-Use Vessel (left) and DASbox Mini Bioreactor (right)
BioFlo® 320: Smart Solution for Bioprocess Control> Workflow Solutions for Molecular Biology> Eppendorf HeatSealer: Effective protection against Evaporation> Further Education with Eppendorf
Application NotesProtein Sample Preparation in Eppendorf Tubes® 5.0 mL · Microinjection into Plant Cells
of Etiolated Seedlings Using the Eppendorf InjectMan® 4 · etc.
Dear Readers,Eppendorf is a synonym for user-oriented processes, innovative technologies, and high-quality premium products for liquid handling, cell handling, and sample handling in life sciences laboratories. In this realm, our broad product portfolio from one (manufacturer’s) source remains unparalleled.
In this issue of BioNews, we introduce to you interesting product innovations: the BioFlo® 320, a flexible solution for process control in cell culture and fermentation, two handy HeatSealers for reliable sealing of different plate formats, our CryoCube®
ultra-low temperature freezers as well as the Eppendorf Cell Imaging Consumables for efficient cell growth and precise fluorescence analysis.
Our process-oriented workflow approach facilitates the targeted, time-saving selection of products for important work steps in your laboratory. Following the cell biology workflow of the previous issue, this edition will show you more about our product solutions for key steps in the molecular laboratory.
Beyond the product, Eppendorf has always been committed to offering its customers an added value. In the case of our Cell Culture Consumables, for example, this is a very innovative packaging concept. For the care and maintenance of your pipettes, we offer veritable “Spa and Wellness” options, and for your career enhancement, attrac-tive seminars and webinars are available to you.
As always, Application Notes and the popular prize competition round off this new edition of your BioNews!
Enjoy reading!
Your BioNews editorial team
EDITORIAL · DEAR READERS
(BN 43) JULY 2015 PAGE 1
Your local distributor: www.eppendorf.com/contact Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels
Abstract
In recent years single-use bioreactors gained more and more importance in animal and human cell culture. With the new line of BioBLU f rigid wall, stirred-tank single-use vessels Eppendorf of-fers premium solutions for microbial applications.
In the following case study, reproducible process control was achieved with par-allel operated BioBLU 0.3f single-use and reusable glass vessels, both used in an Eppendorf DASbox® Mini Bioreactor System (Fig. 1).
Fermentation of E. coli K12 led to very comparable results thus proving the tested single-use vessels to be an ap-propriate tool to accelerate microbial process development and shorten time-to-market in all industries related to microbial production processes.
Introduction
Single-use bioreactors are a suitable tool for time and cost effective biopro-cessing. Minimized setup times, elimi-nated cleaning procedures and there-fore reduced labor time can sustainably accelerate bioprocess development. In all biopharmaceutical industries single-use technologies are widely used in mammalian cell culture.
With the new BioBLU f line, specifically designed to meet the needs of fermen-tation, single-use bioreactors make their way to microbial applications in bio-pharma, food, and cosmetics industries.
Microbial applications make specific demands on the bioreactor design and functionality. Fermentation processes need much higher kLa values for proper mass transfer and suitable heating and cooling options as well.
This comparative study investigates the functionality and reliability of a BioBLU 0.3f single-use mini bioreactor and an autoclavable DASbox Mini Bio-reactor (Fig. 2) in a small scale E. coli fermentation.
Materials and methods
E. coli K12 (DSM 498) was cultivated in a fully instrumented Eppendorf BioBLU 0.3f single-use mini bioreactor and compared to fermentations in con-ventional autoclavable glass bioreactors.
The ready-to-use rigid wall stirred-tank single-use bioreactors, specifically designed for microbial applications, are equipped with a 2x Rushton-type impeller, liquid-free Peltier exhaust condensation and direct drive for high performance agitation. The overhead-driven autoclavable DASbox Mini Bio-
reactors included 2x Rushton-type impeller and liquid free Peltier exhaust condensers as well. A 4-fold parallel DASbox Mini Bioreactor System with active heating and cooling capacities was used with DASGIP® Control Software (now DASware® control 5) for precise process control.
Starting with a working volume of 100 mL each, the cultures were grown for 40 h in PAN medium with an initial glucose con-centration of 40 g/L and fed with 50 % glucose solution in the fed batch phase. The temperature was controlled at 37 °C and pH was adjusted to 6.8 via 4 % ammonia solution; the cultures were submerged aerated with a constant rate of 1 vvm (6 sL/h or 0.1 sL/min). The dissolved oxygen was maintained at 30 % with the stirrer speeds ranging from 600 rpm to 2,000 rpm which equals to tip speeds of 0.94 m/s to 3.14 m/s. Exhaust concentrations were measured and corresponding oxygen transfer rates (OTR) were automatically calculated using a DASGIP GA4 exhaust analysis module.
Results and discussion
Highly reproducible OTR values of up to 250 mmol/L/h were observed in the single-use as well as in the glass biore-actors (Fig. 3).
CLAUDIA M. HUETHER-FRANKEN* AND SEBASTIAN KLEEBANK,
Fig. 1: DASbox Mini Bioreactor System for microbial applications equipped with BioBLU 0.3f Single-Use Vessels and autoclavable DASbox Mini Bioreactors with Rushton-type impeller. Fig. 2: BioBLU 0.3f Single-Use Vessel (left) and DASbox Mini Bioreactor (right)
3CONTENTS
CLAUDIA M. HUETHER-FRANKEN, SEBASTIAN KLEEBANK
A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels
1 – 2
DÖRTE POBURSKI, ANNETT MÜLLER, RENÉ THIERBACH, RAFAL GRZESKOWIAK
Protein Sample Preparation in Eppendorf Tubes® 5.0 mL
3 – 4
RICK COHEN
Low O2 Levels in the Galaxy® 170 R CO2 Incubator Enhance the Efficiency of Reprogramming Human Somatic Cells to Pluripotency
5 – 6
TIM KUNKEL
Microinjection into Plant Cells of Etiolated Seedlings Using the Eppendorf InjectMan® 4
7 – 8
IN THE SPOTLIGHT The Smarter Solution: BioFlo® 320 Bioprocess Control Station 4 – 5
STRAIGHT FROM THE LAB Workflow Solutions for Molecular Biology 6 – 7
Effective Protection against Evaporation 11
Further Education: New Courses and Webinars 12
INNOVATION Cell Imaging at Eppendorf 9
CLOSE-UP Smart Packaging for Premium Products 11
NEWS / TIPS Single-Use Systems In Cell Culture and Microbiology 5
Stay Cool! CryoCube® ULT Freezers 8
Spa and Wellness for Your Pipettes 8
“Best New General Lab Product” Multipette® M4 / Repeater® M4 10
Research Prize Winners Visit Eppendorf in Hamburg 13
SERVICE Prize Competition 14
Reply Fax / Readers’ Service 15
8 119
4 IN THE SPOTLIGHT · THE SMARTER SOLUTION: BIOFLO® 320 BIOPROCESS CONTROL STATION
The Smarter Solution: BioFlo® 320 Bioprocess Control Station
Glass or plastic? No problem
The BioFlo 320 bioprocess control station is the solution for evolving cell culture and fermentation processes. The user is free to choose from our comprehensive portfolios of autoclavable and single-use BioBLU® vessels. This includes the BioBLU 5p, the first single-use vessel to utilize the exclusive packed-bed impeller for continuous perfusion processes. Thirty interchangeable autoclavable and single-use vessels means any laboratory can configure, upgrade, and scale their processes to their needs. The BioFlo 320 can handle the demands of the ever-changing laboratory.
From 250 mL – 40 L, one size fits all
The natural evolution of process develop-ment leads to larger volumes. The need to scale up between control platforms as process volume increases is one of the most time-intensive steps in the process development world. With the introduc-tion of the BioFlo 320, the size of the equipment no longer has to “scale up” with the volume of the process. Its com-pact design remains viable even as the process moves from R&D to production.
Simplify with universal sensor connectivity
The BioFlo 320 also utilizes multiple uni-versal ports for digital sensors. While relatively new to the market, digital sen-sors have an advantage against which tra-ditional analog sensors cannot compete.
The BioFlo 320 bioprocess control station is the flexible new solution for cell culture and fermentation. The need
for process flexibility is paramount to maximizing laboratory output. Whether it be early research and develop-
ment or GMP manufacturing, the ability to grow and adapt with a process is crucial when planning for the future.
The BioFlo 320 is equipped with comprehensive features that will enhance laboratory flexibility for years to come.
KEVIN VOLL, EPPENDORF, INC., ENFIELD, USA
The communication to a digital sensor is the same regardless of sensor type. This means that a pH sensor can be discon-nected from the BioFlo 320 and a CO2 sensor can be connected to the same port, completely changing the process capabilities, with no added effort. Analog sensors require dedicated elec-tronics per sensor, making this impossi-ble. This is a decided advantage for those processes which may not yet have a defined control strategy, typically in the research and development stage.
“Future proof” your laboratory with the BioFlo 320. Autoclavable vessels and our comprehensive portfolio of BioBLU Single-Use Vessels provide process flexibility.
Upgradable solutions through modular design
The mystery around modular design lies in the complexity of the integration. While some equipment may claim to be upgradable, the processes involved in making changes to equipment may sometimes outweigh the benefits gained.
Ease of use is the key to modular design. The BioFlo 320 removes this complexity. This is achieved through interchangeable sparge and overlay gas modules.
Tip
Single-Use Systems In Cell Culture and MicrobiologyBenefit now from the advantages of
BioBLU® Single-Use Vessels!
Eppendorf offers a range of adaptor kits
that convert your existing autoclavable
bioreactor system to the use of BioBLU
Single-Use Vessels without the expense
of replacing the whole system.
Adaptor kits are available for Eppendorf
DASbox® and DASGIP® systems, New
Brunswick™ controllers as well as for
units from Sartorius® and Applikon®. The
easy-to-install kits provide all the neces-
sary equipment for conversion.
BioBLU vessels combine the advantages
of single-use technology with the trusted
performance of a stirred-tank design.
The large portfolio of small, bench, and
pilot-scale bioreactors includes single-use
solutions for microbial fermentation, and
suspension and adherent cell cultures.
Benefit from easy handling, minimized
set-up time and the elimination of cleaning
and sterilization steps to shorten your
development times and reduce costs – in
cell culture and microbiology!
For more details contact your local
Eppendorf organization or visit
www.eppendorf.com/bioprocess. You can
also request the respective brochure by
using the reference number denoted below!
5THE SMARTER SOLUTION: BIOFLO® 320 BIOPROCESS CONTROL STATION · IN THE SPOTLIGHT
Created with the user in mind, these modules are easily installed without the need of a service technician. Using a simple drawer design, the modules allow the user to scale up their gas flow rates for increased vessel volume or change between cell culture and fermentation processes. Process scalability and flex-ibility with the same control station are now within reach of the scientist.
Next generation control software
What good is the ability to modify equip-ment if you are still constricted by a lim-ited software platform? Control of various processes from a single piece of equip-ment requires more than just a modular design. The BioFlo 320 removes unnec-essary process limitations by delivering a universal gas control strategy for both microbial and cell culture applications.
Control up to eight units from a single user interface.
Four available front-mounted peristaltic pumps with easy load pump heads
15 inch touchscreen interface simplifies control and provides clear viewing of process parameters.
Unparalleled multi-unit connectivity al-lows for control of up to eight individual processes from a single user interface.
Like the hardware, the BioFlo 320 control software is designed to keep a laboratory prepared for whatever the future brings.
Highly evolved
Designed and developed for flexibility and power, the BioFlo 320 eliminates the need to invest in new equipment with each process change. Combined with a sincere commitment to quality, the BioFlo 320 truly is the premium choice in bench-scale bioprocess control stations.
6 STRAIGHT FROM THE LAB · WORKFLOW SOLUTIONS FOR MOLECULAR BIOLOGY
Workflow Solutions for Molecular Biology
TANJA MUSIOL, EPPENDORF AG
A major focus of molecular biologists is the study of processes at the intracellular level. Mechanisms and processes
are broken down into their individual steps and the components involved in them are analyzed. Very often, nucleic
acids and/or proteins play a crucial role. Their purification and analysis are among the most frequent applications
in the modern molecular biology laboratory. In this report we will introduce to you Eppendorf products for central
work steps.
The sample material is often available in different volumes and/or concentrations as well as in varying qualities. Since purification and analysis are among the first work steps in the entire laboratory process, obtaining reliable data and avoiding loss of valuable sample material are essential in this initial phase. Errors would multiply manifold during down-stream applications.
Process-oriented solutions
Eppendorf’s focus is on the development of process-oriented solutions at a very high quality level. Our instruments ensure most reliable and reproducible experi-
mental parameters for sample processing under consistent conditions. High quality consumables with innovative features such as the Eppendorf LoBind® surfaces offer the best prerequisites for consistent and uncompromised processing, analysis, and storage of valuable samples. Transfer of extremely small volumes places high demands on liquid handling in the labo-ratory. Eppendorf takes care of these demands with its electronic and manual pipettes and automated systems. Tailored accessory solutions, e.g. for centrifugation of spin columns or photometric quantifi-cation of even the smallest volumes, fur-ther ease the daily work in the lab.
Purification
For sample purification of, for example, DNA or proteins, spin columns, magnetic beads, or vacuum processes may be used. Depending on sample throughput, this can be achieved manually or with the help of automated systems.
The Eppendorf epMotion®, an automated liquid handling workstation, enables higher throughput sample purification using a vacuum chamber or magnetic beads. For manual purification, we recom-mend the special Eppendorf Kit rotor®, available for the Centrifuges 5424/5424 R, 5427 R, and 5430/5430 R.
7WORKFLOW SOLUTIONS FOR MOLECULAR BIOLOGY · STRAIGHT FROM THE LAB
epMotion: The automated liquid handling workstation takes care of higher throughput sample purification via vacuum chamber or magnetic beads.
Eppendorf ultralow-temperature freezers offer maximum sample safety and energy efficiency.
The Concentrator plus (U.S./CAN: Vacufuge plus) concen-trates your samples fast, efficiently, and gently.
Eppendorf Kit rotor for centrifugation of spin columns
This rotor features an extended rotor rim which is optimized for centrifugation of spin columns. It supports the lids of the tubes into which the sample eluate is transferred and thus prevents the lids from shearing off during centrifugation.
Quantification
Following sample purification, exact quantification of the sample is necessary. In addition to the concentration, the quality of the sample, i.e. its purity, is also determined in most cases. Depend-ing on whether a complete sample scan or individual wavelengths are required for ratio determination, the Eppendorf BioSpectrometer® family provides the right instrument for every application.
The BioSpectrometer fluorescence, for example, offers the possibility of quanti-fying the sample with utmost specificity and sensitivity using fluorescence dyes. With this method, dsDNA can be quanti-fied reliably to a concentration as low as 1.0 pg/µL.
The Eppendorf µCuvette® G1.0 was de-veloped specifically for measurement of even the smallest volumes (≥ 1.5 µL). It can be used in any Eppendorf BioPhotometer® or BioSpectrometer.
Amplification & analysis
Following purification, amplification of nucleic acids is frequently the next step. Specific sequences are amplified in a highly precise manner using PCR. In or-der to establish a stable, reproducible PCR it is necessary to know the optimum experimental conditions. This is where the gradient cyclers Mastercycler® pro or Mastercycler nexus come on the scene.
Optimizing the temperature for primer annealing can be achieved in a single run using a temperature gradient. This saves valuable time and ensures stable experimental conditions.
Storage
Prior to downstream applications the samples are often stored. This step also requires reliable conditions in order to ensure that the sample is available unal-tered and of good quality for further ex-periments.
For storage purposes Eppendorf offers a large selection of reliable and energy- efficient ultra-low temperature freezers (see page 8).
Before storing the samples, it is also pos-sible to reduce their volume by concen-tration in the Concentrator plus (U.S./CAN: Vacufuge® plus).
To prevent sample loss during storage, Eppendorf Tubes® or Eppendorf Plates®
with LoBind surfaces are a good choice. Sealing of plates using the Eppendorf HeatSealer (see page 11) prevents liquid evaporation as well as contamination of the sample during the storage.
Product information and Application Notes are available at www.eppendorf.com/workflows.
Eppendorf BioSpectrometer fluorescence: combination of UV/Vis and fluorescence measurements in a single device
News
Spa and Wellness for Your PipettesEppendorf pipette service
Your pipette’s precision and accuracy are
important to ensure the quality of your
results. The Eppendorf service experts
provide quick and competent support with
regular maintenance, calibration, and
adjustment to assure your pipettes continue
to generate reproducible results.
Watch our video for more information
Why not take a short break and see for
yourself what a pipette “spa and wellness”
treatment includes? Find more info in our
new video on the internet at
www.eppendorf.com/pipette-video.
Or scan the QR code below to view it on
your smartphone or tablet PC.
Our Pipette Performance Plans* include a
variety of service options: from affordable
quick checks to GLP/GMP custom-designed
calibration services – according to local
and international standards. And not just
for Eppendorf pipettes, but also for other
brand pipettes and dispensers.
Treating your pipettes well is worth it!
You will also profit from our services for
your pipettes:
> Consistent and reliable results
> Highest confidence in results
> GLP compliant audited systems
For more information, service requests,
and local offers please go to
www.eppendorf.com/epServices.
*Performance Plans are available in selected countries only and service offers may differ.
8 NEWS · STAY COOL! CRYOCUBE® ULT FREEZERS
Stay Cool! CryoCube®
ULT Freezers
MARY LISA SASSANO, EPPENDORF, INC., ENFIELD, USA
Ease of use
When a freezer is easy to use, it makes processes more efficient and more con-venient. CryoCube freezers are designed with convenience and comfort in mind. A new automatic vent port located on the front of the CryoCube F570 and F570h improves energy consumption and tem-perature uniformity while providing easier access to the samples.
The CryoCube F570 and F570h have newly-designed, ergonomic handles that can be operated with less force. The new handles allow for easy one-handed oper-ation. Inside the freezer, the inner doors are now equipped with magnetic closures.
The new CryoCube ultra-low temperature freezers are the latest addition
to an already robust Eppendorf freezer portfolio. Consisting of four product
lines, capacities ranging from 101 L – 760 L, and both upright and chest
orientations, Eppendorf freezers ensure that customers can choose the
perfect freezer for their specific need and laboratory space.
The new automatic vent port located within the control panel
NEW! CryoCube F570, F570h, and FC660h ultra-low temperature freezers
These allow simple opening and closing, further improving the user experience.
Energy-saving solutions
CryoCube freezers utilize an improved refrigeration system that includes a new high-efficiency fan, compressor, and condenser. These new enhancements ensure that CryoCube freezers continue a proud Eppendorf tradition of energy efficiency.
The CryoCube F570h and FC660h (high energy efficiency models) further im-prove energy consumption compared to the F570 and FC660. They combine all the enhancements of the F570 and FC660 with additional vacuum insulation panels.
Confidence
The new CryoCube freezers deliver premium performance and dependable quality. Users can relax, and be confident that their samples are safe in CryoCube ultra-low temperature freezers.
Further information
For more details on CryoCube freezers and other Eppendorf freezers, please see the current brochure or visit www.eppendorf.com/freezer.
Video “Eppendorf Pipette Service - Spa and Wellness for Your Pipettes”
CryoCube® ULT Freezers • Ref. no. 284
(BN 43) JULY 2015 PAGE 1
Your local distributor: www.eppendorf.com/contact Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com
A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels
Abstract
In recent years single-use bioreactors have gained more and more importance in animal and human cell culture. With the new line of BioBLU f rigid wall, stirred-tank single-use vessels Eppen-dorf offers premium solutions for mi-crobial applications.
In the following case study, reproducible process control was achieved with par-allel operated BioBLU 0.3f single-use and reusable glass vessels, both used in an Eppendorf DASbox® Mini Bioreactor System (Fig. 1).
Fermentation of E. coli K12 led to very comparable results thus proving the tested single-use vessels to be an ap-propriate tool to accelerate microbial process development and shorten time-to-market in all industries related to microbial production processes.
Introduction
Single-use bioreactors are a suitable tool for time- and cost-effective biopro-cessing. Minimized setup times, elimi-nated cleaning procedures and there-fore reduced labor time can sustainably accelerate bioprocess development. In all biopharmaceutical industries single-use technologies are widely used in mammalian cell culture.
With the new BioBLU f line, specifically designed to meet the needs of fermen-tation, single-use bioreactors make their way to microbial applications in bio-pharma, food, and cosmetics industries.
Microbial applications pose specific demands on the bioreactor design and functionality. Fermentation processes need much higher kLa values for proper mass transfer and suitable heating and cooling options as well.
This comparative study investigates the functionality and reliability of a BioBLU 0.3f single-use mini bioreactor and an autoclavable DASbox Mini Bio-reactor (Fig. 2) in a small scale E. coli fermentation.
Materials and methods
E. coli K12 (DSM 498) was cultivated in a fully instrumented Eppendorf BioBLU 0.3f single-use mini bioreactor and compared to fermentation in con-ventional autoclavable glass bioreactors.
The ready-to-use rigid wall stirred-tank single-use bioreactors, specifically designed for microbial applications, are equipped with a 2x Rushton-type impeller, liquid-free Peltier exhaust condensation and direct drive for high performance agitation. The overhead-driven autoclavable DASbox Mini Bio-
reactors included 2x Rushton-type impeller and liquid-free Peltier exhaust condensers as well. A 4-fold parallel DASbox Mini Bioreactor System with active heating and cooling capacities was used with DASGIP® Control Software (now DASware® control 5) for precise process control.
Starting with a working volume of 100 mL each, the cultures were grown for 40 h in PAN medium with an initial glucose con-centration of 40 g/L and fed with 50 % glucose solution in the fed batch phase. The temperature was controlled at 37 °C and pH was adjusted to 6.8 via 4 % ammonia solution; the cultures were submerged aerated at a constant rate of 1 vvm (6 sL/h or 0.1 sL/min). Dis-solved oxygen was maintained at 30 % with the stirrer speeds ranging from 600 rpm to 2,000 rpm which equals to tip speeds of 0.94 m/s to 3.14 m/s. Exhaust concentrations were measured and corresponding oxygen transfer rates (OTR) were automatically calculated using a DASGIP GA4 exhaust analysis module.
Results and discussion
Highly reproducible OTR values of up to 250 mmol/L/h were observed in the single-use as well as in the glass biore-actors (Fig. 3).
CLAUDIA M. HUETHER-FRANKEN* AND SEBASTIAN KLEEBANK,
Fig. 1: DASbox Mini Bioreactor System for microbial applications equipped with BioBLU 0.3f Single-Use Vessels and autoclavable DASbox Mini Bioreactors with Rushton-type impeller. Fig. 2: BioBLU 0.3f Single-Use Vessel (left) and DASbox Mini Bioreactor (right)
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A Comparative Study: Small Scale E. coli Cultivation Using BioBLU® Single-Use and Reusable Vessels
Supporting kLa values of up to 2,500 h-1 were determined by static sulfite deple-tion method (data not shown) demon-strating that the single-use design of the BioBLU 0.3f bioreactors perfectly matches the demands of microbial applications.
Biomass production was determined offline as cell wet weight and revealed comparable growth characteristics in single-use and glass mini bioreactors (Fig. 4).
The maximal biomasses of about 160 g/L achieved in the fermentation are equal to an OD600 of about 100 (data not shown).
Conclusion
This case study demonstrates that the BioBLU 0.3f single-use bioreactor ad-dresses the specific needs of E. coli fermentation especially in regard to
mass and heat transfer. OTR values measured in the single-use vessel pro-cess runs as well as the final biomasses reached were comparable to those achieved with the conventional auto-clavable DASbox glass bioreactors.
The specifically adapted single-use de-sign of the BioBLU 0.3f mini bioreactor supports the high demands of microbial applications. Used with the Eppendorf DASbox this single-use bioreactor is a premium tool for screening, media opti-mization, and use as a scale down model for process development including De-sign of Experiments (DoE) approaches.
Industry interest in adequate single-use bioreactor solutions for fermenta-tion is steadily increasing. With the BioBLU f line of single-use vessels Eppendorf offers premium solutions for microbial applications. Users in fermen-tation can now benefit from advanced
process control, accelerated process development, reduced costs, and shorter time-to-market.
Fig. 3: Reproducibility of online calculated oxygen transfer rates (OTR) of parallel process runs at constant temperature of 37 °C using BioBLU 0.3f single-use and reusable DASbox Mini Bioreactors, respectively.
Fig. 4: Biomass production. Cell wet weight of fermentations carried out in BioBLU 0.3f single-use and reusable DASbox Mini Bioreactors, respectively.
Readers’ serviceBioBLU® f • Ref. no. 279
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Protein Sample Preparation in Eppendorf Tubes® 5.0 mL
Abstract
Analysis of proteins from cultured cells often requires sample processing at a larger volume scale. Examples include isolation of cell fractions from homog-enates, determination of enzyme activi-ties as well as harvesting of proteins from sparsely growing cells. The new Eppendorf Tubes 5.0 mL are the ideal vessel format for these applications. Their use allows larger processing vol-umes, high centrifugation forces, and improved handling.
Introduction
Over the past decades reaction tubes in the microliter range have become the mainstay in the laboratory with volume formats of 0.5 mL, 1.5 mL, and 2.0 mL used most frequently. In the field of protein isolation and analysis, however, many steps require sample processing at a larger volume scale: isolation of cell fractions or proteins from sparsely growing cells, determination of enzyme activities, or immunoblot of protein isolates.
The new Eppendorf Tubes 5.0 mL are ideally suited for such applications: their use allows large processing volumes
and high centrifugation forces as well as easy and convenient single-handed operation.
In this Application Note the performance of the Eppendorf Tube 5.0 mL during protein sample preparation was investi-gated. The following questions were addressed:
1) Are the required steps easily adapt-able to the new format and do tempera-ture differences during processing of larger volumes present a problem?
2) Does the use of different tube for-mats lead to quantitative differences (e.g. caused by sample loss)?
3) Does the use of the larger tube format result in compromised protein quality or activity?
To investigate these questions, com-parative protein preparation was per-formed in different tube formats.
Materials and methods
Comparative cell lysate preparation
Human cells (HT29) were grown to a confluence of ca. 70 % and harvested in 600 μL lysis buffer (Cell Signaling Technology®, Inc.).
DÖRTE POBURSKI, ANNETT MÜLLER AND RENÉ THIERBACH,
FRIEDRICH-SCHILLER-UNIVERSITÄT, INSTITUTE OF NUTRITIONAL SCIENCE, JENA, GERMANY
RAFAL GRZESKOWIAK, EPPENDORF AG, HAMBURG, GERMANY
The pooled cell suspensions were ad-justed with lysis buffer to 20 mL and pipetted to three tube formats:
Eppendorf Safe-Lock Tubes 1.5 mL, 2.0 mL, and Eppendorf Tubes 5.0 mL. The samples were snap-frozen in liquid nitrogen* and stored over night at −80 °C. After thawing on ice, samples were homogenized by sonication and the membrane debris was removed by centrifugation (3,200 x g, 4 °C, 10 min).
The supernatants were transferred to new reaction tubes. Total protein quan-tification was done using BCA method (absorbance at 562 nm measured in a plate reader). The samples were subse-quently mixed with Laemmli buffer and analyzed via SDS PAGE and immuno- blot method.
Determination of aconitase activity
Murine embryo fibroblasts were grown to a confluence of ca. 50 % and harvest-ed in 600 μL of Tris buffer (50 mM). The samples were transferred to 5.0 mL or 1.5 mL Eppendorf Tubes, resuspended and snap-frozen in liquid nitrogen*.
Following storage at −80 °C the sam-ples were thawed on ice and sonicated; the cell debris was removed by centrifu-gation. The supernatant was standard-ized to 1.1 μg/μL of total protein using Bradford method and aliquots (130 μg of total protein) were diluted to 150 μL with Tris buffer. After addition of 150 μL of assay buffer (50 mM Tris, 60 mM sodium citrate, 1 mM MnCl2, 0.4 mM NADP+), formation of NADPH was measured in a plate reader (37 °C, 340 nm) and the relative aconitase activity was calculated accordingly.
Results and discussion
Isolation and processing of protein samples pose high requirements on tubes used during this procedure: broad temperature range, high centrif-ugal forces, tight lid closure, and easy handling.
In this application we compared the use of different Eppendorf tube for-mats (1.5 mL, 2.0 mL, and 5.0 mL) for protein preparation from cell lysates.
Eppendorf Safe-Lock Tubes 1.5 mL
Eppendorf Safe-Lock Tubes 2.0 mL
Eppendorf Tubes 5.0 mL
Filling volume with cell suspension (approx. 2/3 of the maximum volume)
1.0 mL 1.3 mL 3.3 mL
Time until thawed on ice 60 min 70 min 90 min
Volume transferred following cen-trifugation 0.9 mL 1.2 mL 3.0 mL
Addition of Laemmli buffer 0.45 mL 0.6 mL 1.5 mL
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Protein Sample Preparation in Eppendorf Tubes® 5.0 mL
In Table 1 the main sample processing steps are compared.
The 5.0 mL tubes provided several ad-vantages as compared to smaller tube formats: optimized homogenization with a sonicator, minimization of material spillage and sample loss by adhesion to the sonication rod.
During subsequent centrifugation the Eppendorf Tubes 5.0 mL also displayed clear advantages over the smaller tubes: Various rotors and accessories available for the Eppendorf Tubes 5.0 mL allow easy fit with most centrifuges and ap-plication of high centrifugal forces to obtain clear lysate (up to 25,000 x g).
The handling, too, was improved: Re-moving the supernatant was prone to less contamination and faster.
Along with all mentioned advantages the quality of the cell lysate prepared in the Eppendorf Tubes 5.0 mL remained unchanged. As shown in Fig. 1, no sig-nificant differences in protein concen-trations between different vessel for-mats could be detected. These data show that processing larger volumes in the 5.0 mL format does not lead to a quantitative sample loss.
Furthermore, in order to investigate whether using the 5.0 mL format might compromise protein quality a compari-
son of enzymatic activity (aconitase) was performed in cell lysates pro-cessed in 1.5 mL and 5.0 mL tubes. No differences could be detected in enzyme preparations when compared between 1.5 mL and 5.0 mL tubes (data not shown).
This demonstrates that the processing of samples in Eppendorf Tubes 5.0 mL influences neither the quality nor the activity of proteins in the tested cell lysates.
Conclusion
The use of the new Eppendorf Tubes 5.0 mL for processing of larger protein sample volumes allows considerable handling optimization of essential workflow steps compared to smaller tube formats. The new tubes can be easily employed for volumes between 1.0 mL and 5.0 mL. Their larger conical shape assures thorough homogenization and minimization of sample spillage as well as optimal handling: Removing the supernatant was prone to less contami-nation, and it was faster.
The experiments have shown that all steps required for sample preparation can be easily adapted to the new tubes and that differences in temperature ad-justment do not pose a problem during sample processing. Furthermore, neither quantitative nor qualitative deteriora-tion of the proteins obtained could be detected.
Pro
tein
con
cen
trat
ion
[µg
/µL
]
Total protein quantification
Eppendorf Tubes 1.5 mL
Eppendorf Tubes 2.0 mL
Eppendorf Tubes 5.0 mL
1.2
0.8
0.4
0.0
Fig. 1: Total protein quantification using BCA method of cell suspensions processed in the different Eppendorf tube formats. The results obtained with preparations in different formats showed no significant differences (t-test).
Readers’ serviceEppendorf Tubes® 5.0 mL • Ref. no. 264
*Safety notice: operating temperature for Eppendorf Tubes 5.0 mL is − 86 °C to + 100 °C. Work with liquid nitrogen is at user’s own risk. For details refer to the operating manual.
Table 1: Comparison of sample processing steps for immunoblot analysis using different Eppendorf tube formats: 1.5 mL, 2.0 mL, and 5.0 mL
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Low O2 Levels in the Galaxy® 170 R CO2 Incubator Enhance the Efficiency of Reprogramming Human Somatic Cells to Pluripotency
Abstract
In order to successfully reprogram hu-man somatic cells to the pluripotent state, several variables must be consid-ered and optimized. One of the most overlooked variables is the atmospheric composition in which the cells are cultured, reprogrammed, and expand-ed as induced Pluripotent Stem Cells (iPSCs). We generated iPSCs in either standard norm-oxy conditions or under low O2 (4 %) conditions in the Galaxy®
170 R CO2 incubator.
While both conditions were permissive for iPSC generation, the low O2 condition allowed earlier detection of colonies, more mature looking colonies, and a larger and more robust number of colo-nies by 21 days post-electroporation. Controlled O2 conditions should be a vital part of optimized culture systems to generate iPSCs. Galaxy incubators with optional O2 control provide stable O2 levels in a range of 0.1 – 19 % and an optimized environment.
Introduction
In hopes of using iPSCs as a starting material for cell-based therapeutics, there is an ongoing search for optimal types of patient starting materials, de-fined media, and methods of culture that do not induce genetic modification. A key element in developing robust cul-tures is the atmosphere in which cells are generated and developed. Shinya Yamanaka [1] demonstrated early on that hypoxic conditions (low O2 condi-tions) enhance cellular reprogramming. Since then, the majority of publications involving iPSCs continue to use ambi-ent O2 concentrations with 5 % carbon dioxide (CO2) as the norm. In this study, we report an optimal reprogramming protocol for human foreskin fibroblasts using electroporation of a single episom-al vector cultured in a small molecule cocktail-containing media.
Materials and methods
Shortened method section; for details on products, plasmid construction, and me-dia composition, see Application Note 338 at www.eppendorf.com/application.
RICK COHEN, RUTGERS UNIVERSITY, PISCATAWAY, NJ, USA
A more detailed stem cell methodology handbook is available by email request from [email protected].
Fibroblast culture
Human foreskin fibroblasts were cul-tured on 10 cm2 uncoated TC-treated dishes in norm-oxy conditions in com-plete FibroLife® Serum Free medium (Lifeline Cell Technology®) containing 2 % HI-FBS and 1 % Pen/Strep. Routine expansion until passage 9 was carried out using TrypLE™ (Life Technologies®) for cell dissociation.
iPSC induction
Prior to electroporation procedures, target 6-well plates (one for norm-oxy, and two for low O2 conditions) were coated with Matrigel® for 1 h at 37 °C, and then pre-equilibrated with Repro-gramming Media Step 1.
The pellet was resuspended in electro-poration solution to a density of 1.1 x 106 cells per 100 µL. The cells were mixed in duplicate with 5.5 µg (10 µL) of low endotoxin preparation of pERC-V1 DNA,
and then electroporated at 1,700 V, 10 ms, 1 pulse. The day after electroporation, the media was changed to Reprogram-ming Medium Step 2. This media was refreshed every other day until colonies were visibly distinguished, then changed daily until passaging.
At 21 days post-electroporation, clearly passable colonies were seen in the low O2 cultures whereas smaller, less abun-dant clones were available in the norm-oxy conditions. Clones were picked and grown in various conditions as de-scribed below. Once the primary se-lected colonies were of appropriate size, the cultures of presumptive iPSCs were selected and passaged.
Fluorescent marker staining
Following 5 to 6 passages, cells were plated onto Matrigel-coated 24-well culture plates and grown to sub-conflu-ent density whereafter they were fixed and stained with 3 stem cell markers. Samples were counterstained with the DNA marker, DAPI, and visualized us-ing standard microscopy techniques.
Hypoxic conditions
12 d
ays
21 d
ays
Norm-oxy conditions
Fig. 1: Generation of iPSCs under varying O2 concentrations in the Galaxy 170 R incubator. After 12 days, early colony formation is seen in low O2 cultures, whereas this is not observed as early in norm-oxy cultures. By 21 days post-electro-poration, clear colonies are seen in both, however the size and number are more numerous in low O2 cultures.
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Results and discussion
The day after electroporation, fibro-blast cultures from low O2 and norm-oxy conditions were observed for RFP fluorescence. Roughly 40 % of the cells appeared to express RFP brightly. During the first several days of blastici-din selection (for plasmid details see Application Note), there was cell death of many non-transfected and some RFP-expressing cells.
By 10 – 12 days (Fig. 1), changes in the morphology of the normally spindle- shaped fibroblasts were observed as many cells in the low O2 conditions be-gan to become cuboidal in shape and displayed a more compact cytoplasm. Fewer cells, if any, were seen during this time period in the norm-oxy condi-tions. By 15 – 16 days, morphological changes were very clear in the low O2 cultures, and these alterations were starting to become apparent in the norm-oxy conditions.
By 21 days post-electroporation, dozens of robustly growing colonies were in all 3 cell lines assayed. Whereas under norm-oxy conditions, less than 5 colo-nies were observed, and when present, often they were 1/3 or less in size, com-pared to the low O2 counterparts (Fig. 1).
Low O2 Levels in the Galaxy® 170 R CO2 Incubator Enhance the Efficiency of Reprogramming Human Somatic Cells to Pluripotency
Low O2 conditions are far more favor-able for generating iPSCs.
It was evident that initial steps to select a stable cell line were more easily ac-complished in low O2 conditions. To ex-pand growing colonies, the cells were mechanically picked, with or without prior Dispase treatment, into fresh medium and transferred onto fresh Matrigel-coated 6-well plates. In low O2 conditions, many of the colonies at-tached well, and grew robustly, to form iPSC colonies with typical morphology (Fig. 2).
In norm-oxy conditions, many colonies had mixed morphological cell types. Af-ter 3 selective passages, seemingly sta-ble iPSCs were obtained from norm-oxy conditions.
After 4 to 5 passages, successfully re-programmed colonies from both atmo-spheric conditions were seeded for standard indirect immunostaining tech-niques. Colonies replated after Dispase treatment tended to attach better than those that were mechanically picked. When treated with Y-27632, the colo-nies survived the process at a higher rate and formed colonies within 4 days. By 7 days post-passaging, many colo-nies were observed.
The morphology observed with low O2 cultures resembled the more purified mature iPSC colonies. Triple staining of cultures revealed that low O2 conditions were favorable for the expansion and development of iPSC colonies with sta-ble typical morphology. Similar staining was observed in norm-oxy conditions.
Conclusion
The Galaxy 170 R with optional O2 con-trol was able to increase the efficiency of the generation of stable iPSC lines using a non-optimized and novel reagent. Without the regulation of O2, this method yielded marginal results and could have been discarded as inefficient. We were consistently able to observe morphologi-cal changes in fibroblast cultures culti-vated in low O2 conditions within 12 days post-electroporation, and clearly robust colonies after 3 weeks. The advantage of maintaining a low O2 environment during reprogramming is clear.
Literature
[1] Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 2009; 5(3):237–41.
Norm-oxy conditions Hypoxic conditions
Readers’ serviceGalaxy® 170 R • Ref. no. 273
Fig. 2: Primary passaging of pERC-V1 reprogrammed iPSCs. Colonies replated after Dispase treatment tended to attach better than those mechanically picked. When treated with Y-27632, colonies survived the process more efficiently and formed colonies within 4 days. By 7 days post-passaging, many colonies were ob-served, however the morphology observed with low O2 cultures tended to resemble more purified mature iPSC colonies.
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Microinjection into Plant Cells of Etiolated Seedlings Using the Eppendorf InjectMan® 4
TIM KUNKEL, FACULTY OF BIOLOGY, ALBERT-LUDWIGS-UNIVERSITY FREIBURG, GERMANY
Abstract
For several model systems microinjec-tion is an established method to intro-duce DNA into single cells to generate transient and stable transformants. Not only nucleic acids but also proteins and small effectors can be injected. The in-jection of plants is however difficult and only a low percentage of the injected cells survive. The efficiency is largely dependent on the equipment used and injection conditions. For this application note the new Eppendorf micromanipu-lation system was used to inject tomato, mustard and Arabidopsis seedlings.
Injections with Lucifer yellow (ly) dye and Alexa Fluor® 488-labeled histone (histone-AF488) protein resulted in a survival rate of approximately 25 % of all cells, 24 h after injection. The new Eppendorf micromanipulation system with its exceptional fast and precise motor movements is apparently very well suited for the injection of plant cells.
Introduction
Microinjection of “higher” plant cells was used several years ago by Chua and co-workers to investigate reactions in response to the light signal cascade [1]. In these investigations cellular re-sponses like plastid development and anthocyanin production could be ob-served after the injection of the red/far red light photoreceptor phytochrome A. Usually plant cells possess a very rigid cell wall that makes microinjection challenging as it requires stable injec-tion capillaries with small diameters.
The cytoplasm surrounding the large central vacuole is only a few µm thick. As a result of the deformation of the cell wall during the injection and the sudden relaxation when the capillary penetrates the wall, the needle will of-ten end up in the vacuole. Material in-jected into the vacuole is not only trapped but in many cases degraded, due to the low pH and the activity of several proteases. Furthermore, be-cause of multiple cell layers, light scat-tering, and pigmentation, the optical properties of whole plant seedlings or
plant organs are not optimal. There-fore, it is often very difficult to identify different cell layers or even subcellular structures like vacuoles under micro-scopic control during the microinjection process.
To approach the described problems, Eppendorf has developed a microma-nipulation system equipped with very fast micromotors, variable injection parameters, high holding and injection pressure in combination with a newly developed piezo step function. The setup described below was tested successful-ly for its suitability to inject plant cells.
Methods
Preparation
Seedlings were placed on wet filter pa-per and incubated for various times in continuous darkness (dD) after germi-nation (Sinapis alba 4 dD, Lycopersicon esculentum 6 dD, Arabidopsis thaliana 4 dD). Germination of Arabidopsis seed-lings was induced by 2 days of cold treat-ment (4 °C) and subsequent irradiation with white light for 6 h.
Microinjection was carried out in a custom-made laminar flow hood on a stone desk to block vibrations from the ventilator.
Microinjection
The microinjection workstation was assembled as follows: the Eppendorf micromanipulator InjectMan 4 was adapt-ed to a Zeiss® Axiovert® 135 inverted microscope (Zeiss, Germany). The piezo actuator of the Eppendorf PiezoXpert®
was mounted onto the motors of the InjectMan 4 at an angle of 35° and its control unit was electronically connect-ed to the InjectMan 4 allowing synchro-nized piezo-assisted injection steps.
The injection pressure parameters were adjusted with the Eppendorf FemtoJet®. The settings of injection parameters are listed in Table 1. The injection pressure was triggered after successful penetra-tion of the cell wall. The total setup is shown in Fig. 1.
Results and discussion
The injection angle was adjusted to 35° and axial injection movements were performed at a speed of 3,000 µm/s with a step size of 10 – 20 µm.
These settings ensure minimal injuries of the cell wall and thus probably raise the survival rate after injection. As a marker for injection Lucifer yellow, a water soluble dye that easily allows dis-crimination between cytoplasmic and
Fig. 1: Overview over the microinjection setup (A) and fixation of etiolated Arabidopsis seedlings (B). (A) From left to right: Eppendorf PiezoXpert controller and piezo actuator mounted to the micromotors, Eppendorf InjectMan 4 with micromotors mounted to an inverse microscope, Eppendorf FemtoJet. (B) Seedlings were fixed with adhesive tape onto a lid of a petri dish and the roots were covered with wet filter paper.
A B
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Microinjection into Plant Cells of Etiolated Seedlings Using the Eppendorf InjectMan® 4
vacuolar localized fluorescence, was in-jected as indicated (Table 1).
Analysis was carried out using a Zeiss YFP or GFP fluorescence filter set, 24 h after injection. For these tests tomato, mustard, and Arabidopsis seedlings were used. In the case of tomato and mustard seedlings injection efficiencies of 26 % and 48 % were obtained (Fig. 2). The injection efficiency for Arabidopsis seedlings was about 10 %, 24 h after injection. Compared to tomato and mus-tard seedlings the injection efficiency is strongly decreased in Arabidopsis seed-lings.
However, these seedlings are much smaller and very fragile. As a conse-quence, injected cells do not only die relatively often but in many cases the whole hypocotyl is affected. Consider-ing these challenges 10 % injection efficiency reflects a good value for Arabidopsis seedlings in our opinion.
Localized fluorescence of Lucifer yellow (Iy) 24 h after injection indicated that the cells were viable, but ly is no in vivo marker. Hence, we decided to perform cytoplasmic injection of Alexa Fluor 488-labeled histone (histone-AF488) as the active import of histone-AF488 into
the nucleus is only possible in living cells [2]. As shown in Table 1 the per-centage of cells showing histone-AF488 fluorescence in the nucleus was in the same range as the observed percentage after ly injection, confirming that these cells survived the injection procedure.
In summary the new Eppendorf micro-manipulation system with its exceptional fast and precise motor movements is apparently very well suited for the in-jection of plant cells. This also includes the injection of Arabidopsis seedlings with relatively high efficiencies.
Since a large collection of Arabidopsis mutants and transgenic lines is available for many research areas, microinjection of Arabidopsis cells from specific organs and tissues should, in addition to injec-tion of other plant species, help to ana-lyze numerous scientific questions.
Acknowledgement
I thank Dr. Stefan Kircher and Prof. Eberhard Schäfer for their support and scientific discussions.
Literature
[1] Neuhaus G, Bowler C, Kern R, Chua NH. Calcium/Calmodulin-dependent and -independent phytochrome signal transduction pathways. Cell 1993; 73:937-952.
[2] Melchior F, Paschal B, Evans J and Gerace L. Inhibition of nuclear protein import by nonhydro-lyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential trans-port factor. J Cell Biol 1993; 123:1649-1659.
The complete version of this Application Note (No. 346) can be downloaded in PDF format at www.eppendorf.com/applications.
Seedling Setting of injection parameters Capillary opening µm
Solution injected
Cells injected Positive cellsPi (hPa) Ti (s) Pc (hPa) No. % (No.)
Table 1: Microinjection of Lucifer yellow (ly) and Alexa Fluor 488-labeled histone (Histone) into etiolated seedlings from tomato, mustard and Arabidopsis. Holding pressure prior to injection (Pc), Injection pressure (Pi), injection time (Ti).
Fig. 2: Fluorescence analyses 24 h after injection into etiolated seedlings of tomato (A), Arabidopsis (B, D) and mustard (C). Lucifer yellow (ly); Alexa Fluor 488-labeled histone (Histone-AF488).
9
Cell Imaging at EppendorfDANIEL WEHRHAHN, EPPENDORF AG
CELL IMAGING AT EPPENDORF · INNOVATION
Cell imaging techniques are used by an increasing number of scientists to gain insights into cellular and tissue
function. Rapid advances in fluorescent protein and synthetic fluorophore technology have made cell imaging a
powerful analytical tool in most cell biology laboratories. In living cells, fluorescent proteins are most commonly
utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments. Fluores-
cence based imaging of cells is achieved with a variety of techniques, including widefield, confocal, and multiphoton
microscopy.
The quality of a cell imaging experiment is mainly influenced by the culture condi-tions, the sample preparation, microscope setup, and image acquisition parameters. The optical quality of the imaging con-sumables is also crucial for a good result.
Whether inverse microscopy of living or fixed cells is used in parallel experi-ments or single investigations, the new Eppendorf Cell Imaging Consumables offer reliable optical performance.
Premium design and manufacturing is combined with vigorous quality assur-ance during production. All Eppendorf cell imaging products are equipped with advanced surface properties to support reliable adhesion and growth of the cells.
Eppendorf Cell Imaging Plates
These are black 24- or 96-well plates with clear bottom made of either a thin 25 μm film or a 170 μm cover glass. The
plates with film bottom show excellent light transmission rates even for UV-A and UV-B light. The autofluorescence of the material is lower than a conventional polystyrene bot-tom with a significant reduc-tion in background signaling. The film bottom enables high gas transfer through the plate bottom while the glass bottom plates offer excellent planarity for sophisticated microscopic analysis.
Eppendorf Cell Imaging Dishes
These dishes ensure excellent results in the high resolution microscopy of living and fixed cells.
The bottom of the 35 mm dishes is made from cover glass. The 2 mm lower, 18 mm wide central cavity with glass bottom enables concentration of the cells on the glass surface during seeding. This also helps to reduce costs of antibodies and dyes. The handling and orientation of the dishes is convenient and safe due to a polygonal gripping zone and orientation marks.
Eppendorf Cell Imaging Slides and Coverglasses
These products offer a high chemical resistance of materials for reliable and reproducible results in fixation protocols. The tool-free removal of chambers from Cell Imaging Slides and Coverglasses makes working easy and convenient. (Use the QR code to watch this in our video!)
Products are available with one to eight chambers depending on your application needs.
NEW! Cell Imaging Consumables
Find the right product for your imaging application at www.eppendorf.com/cic.
Video “Simplified Handling of Imaging Consumables”
Eppendorf Cell Imaging Consumables • Ref. no. 282
Eppendorf Cell Imaging Consumables allow for efficient cell growth and precise fluorescence analysis. NIH 3T3 cells: F-actin staining with phalloidin (red) and nuclei with DAPI (blue)
About the SelectScience® Scientists’
Choice Awards:
SelectScience, an independent, expert-led
scientific review resource for the world-
wide scientific community, began the
Scientists’ Choice Awards in 2007 to
enable scientists to voice their opinions on
the best laboratory products.
Once a year, SelectScience invites mem-
bers to nominate their favorite products
of the year in each category. For more
information, visit
www.scientistschoiceawards.com.
10
“Best New General Lab Product” Multipette® M4/Repeater® M4
CHRISTIANE MARKAU, EPPENDORF AG
NEWS · “BEST NEW GENERAL LAB PRODUCT” MULTIPETTE® M4 / REPEATER® M4
Scientists around the world voted Eppendorf’s Multipette M4 (U.S./CAN: Repeater M4) to be the “Best New
General Lab Product” at the “Scientists’ Choice Awards 2015” organized by the online portal SelectScience®. The
Scientists’ Choice Awards 2015 celebrate “new products that have significantly contributed towards laboratory
efforts in 2014”.
Watch Lars Borrmann showing how the Multipette M4 helps scientists in their daily routine!
The Multipette M4 is a hand-held preci-sion instrument, designed to minimize the time and effort required for precise and highly accurate pipetting or dispens-ing in long series, such as tube or plate filling, aliquoting reagents, and kit usage.
The Multipette M4 can dispense volumes from 1 µL to 10 mL. The aspirated liquid can be dispensed in up to 100 steps without refilling the tip – an ergonomic and fast method to fill a 96-well plate.
An integral step counter displays the number of steps executed, allowing the user to resume with confidence should there be any interruption while dispens-ing.
Versatility, safety, maximum accuracy
Unlike air-cushion pipettes, the Multipette and the syringe-like Combitips advanced®
form a positive-displacement system. This allows precise and accurate dispensing of liquids that are difficult to handle, e.g. viscous or foaming solutions or liquids with high vapor pressure. The system also enables contamination-free dispens-ing of toxic, radioactive, or other hazard-ous liquids.
Award winners
Eppendorf has already won many interna-tional prizes for innovative, high-quality laboratory products, including several Scientists’ Choice Awards:
Category “Best New General Lab Product”
2013: Eppendorf Xplorer® plus 2011: Eppendorf Xplorer 2010: Centrifuge 5430 R
Category “Best New Life Science Product”
2009: Mastercycler® pro
With the 2015 award this tradition is being continued. “It is a great honor that this is a prize that we received based on the vote of customers. We put a lot of passion and effort into developing our products, with a focus on the customer needs”, said Dr. Lars Borrmann, Market-ing Director at Eppendorf North America, Inc., who accepted the prize on behalf of Eppendorf at the Pittcon® 2015 in New Orleans, LA.
“A vote received by the customer is a great honor because it shows that what we do is really well received by the customers.”
Multipette® M4 / Repeater® M4 • Ref. no. 268
Winner of the Scientists’ Choice Award 2015 in the category “Best New General Lab Product”: Multipette M4/Repeater M4
More information: www.eppendorf.com/multipettem4 www.eppendorf.com/repeaterm4
Close-up
Smart Packaging for Premium ProductsEppendorf Cell Culture Consumables meet
the highest requirements for excellent cell
growth, reliability, and protection against
contamination – thanks to a perfect
interplay of product, performance, and
packaging.
Cell culture dishes, for example, are the
format of choice whenever you need direct
access to your cells. During development
we specifically focused on improved
handling. Our efforts resulted in a corru-
gated handling ring and a so-called Splash-
Protect™ Ring inside the dish lid that provide
unsurpassed safety during transportation
and expansion of cells.
Innovative packaging concept
Since the packaging significantly contrib-
utes to product safety and contamination
protection we developed a completely
new concept. The innovative packaging
combines convenient tool-free opening
and secure tape-free closing and an easy
solution to shrink the package for space-
saving, secure, and contamination-free
storage.
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Effective Protection against Evaporation
Every laboratory employs different types of plates. In order to obtain reli-
able assay results, plates need to be sealed tightly and in a reproducible
manner. The same is true for sample storage. Especially for long term
storage, a tight seal is very beneficial to ensure your sample is securely
stored until you need it again.
EFFECTIVE PROTECTION AGAINST EVAPORATION · STRAIGHT FROM THE LAB
KAY KÖRNER, EPPENDORF AG
Introducing the new HeatSealer family, Eppendorf now offers you two small and flexible instruments for reliable sealing of different plate formats: the small, ex-tremely user friendly HeatSealer S100 and the slightly larger HeatSealer S200 with flexibly programmable sealing pa-rameters. Both models ensure an easy, reproducible, and effective plate seal, offering effective protection against evaporation and contamination.
A selection of adapters allows you to work with the most common plate formats. Whether the goal is a hermetic seal of multiwell plates or effective protection
against evaporation in low-profile PCR plates, the Eppendorf HeatSealers are the right choice. The integrated thermostat prevents overheating while simultaneous-ly providing a reliable and reproducible plate seal. The improved contact pressure mechanics ensure even easier handling.
In addition, the HeatSealer S200 allows you to choose from different sealing temperatures and times, depending on the requirements, plate and sealing op-tion.
Watch the video to see how it works!
For more details on Eppendorf Cell Culture Consumables, please request our brochure or visit www.eppendorf.com/ccc.
Eppendorf HeatSealer • Ref. no. 285
Especially small and easy to operate: Eppendorf HeatSealer S100. Pre-programmed sealing temperature of 170 °C.
Especially flexible: Eppendorf HeatSealer S200. Free choice of sealing temperature between 125°C and 200°C in 1 °C steps. Sealing time: 1 to 9 seconds.
Eppendorf Cell Culture Consumables • Ref. no. 270
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Further Education: New Courses and Webinars
BERRIT HOFF, EPPENDORF AG
STRAIGHT FROM THE LAB · FURTHER EDUCATION: NEW COURSES AND WEBINARS
Eppendorf is continually expanding the educational portfolio for its customers. In addition to the classroom
courses at the Eppendorf Training Center (ETC) with intensive hands-on components, the number of webinars
held is being increased in order to reach a larger audience around the world. Dr. Jochen Mueller-Ibeler (Executive
Director Field Applications, Eppendorf AG) and Dr. Jessica Wagener (Field Application Specialist Cell Handling,
Eppendorf AG) report on the latest developments.
“The ETC has been offering seminars for further education in the laboratory since 1997”, states Jochen Mueller-Ibeler. “In these classroom training sessions participants have the opportunity to learn new techniques in theory and practice, refresh knowledge and prepare well for the introduction of new methods.”
New introductory course for cell culture
The new seminar Cell culture basics in theory and practice is offered at the sites in Hamburg (Germany) and Namur (Bel-gium). This 2-day course is designed for technical and scientific personnel with no or little experience in cell culture. “The cultivation of cells poses many challenges. The best way of preventing contamination is by adhering to aseptic techniques”, explains tutor Jessica Wagener. “In our course, the participants learn the basics for successful work in the cell culture laboratory.”
In the theoretical part, the participants learn the basic biology of eukaryotic cells,
the basics of cell culture, and how to prevent contamination. In the practical part, they work under sterile conditions at the biological safety cabinet. These exercises include the storage and sub-cultivation of cells as well as the deter-mination of viability and cell number. “Each participant is provided with his /her own work space at the biological safety cabinet”, concludes Jessica Wagener.
Webinars – a flexible addition
“Webinars supplement the existing courses available at the ETC and reach a much larger audience”, states Mueller-Ibeler. “One example is the webinar Preventing Contamination in Cell Culture which took place on March 31, 2015, moderated by SelectScience®.”
“A wide range of users in the field of cell culture followed this webinar. Contami-nation is a common problem in cell cul-ture; it costs time and money. That’s why the main focus of this webinar was on sterile work techniques and contamina-
tion prevention and on recognizing and identifying the various contaminants”, reports Jessica Wagener. “The feedback was very positive. The participants of the live webinar submitted several questions – most of which I replied to at the end of the webinar. More complex questions were answered individually by email. This shows that webinars offer many opportunities for interaction with the participants. The flexibility of webinars is a major benefit; if someone does not have the time to follow the live session, they can follow the recording.”
According to Mueller-Ibeler, his team will continue to increase the number of webi-nars offered and thus expand the portfo-lio of the Eppendorf Training Center. “Webinars and seminars have ONE goal. We want to address current and relevant topics and demonstrate solutions for the daily workflow in the laboratory!”
Additional information
> Overview of ETC courses: www.eppendorf.com/etc
> Webinars are announced online or in local Eppendorf newsletters. Subscribe now to your newsletter in the myEppendorf section of your local Eppendorf website.
> Webinar Preventing Contamination in Cell Culture: www.eppendorf.com/webinar_pccc
Eppendorf Training Center seminars: Tutors and participants work in small groups in modern labora-tories. Intensive practical sessions ensure optimum learning success.
13
Research Prize Winners Visit Eppendorf in Hamburg
BERRIT HOFF UND CAROLYN TAUBERT, EPPENDORF AG
RESEARCH PRIZE WINNERS VISIT EPPENDORF IN HAMBURG · NEWS
Successful research on bats
Michael Yartsev, Ph. D., CV Starr Postdoc at the Princeton Neuroscience Institute and 2013 winner of the Eppendorf & Science Prize for Neurobiology visited Eppendorf in Hamburg in July 2014 to present his ground-breaking work.
Michael Yartsev uses an unusual animal model, the bat, to study the underlying neural mechanisms of spatial memory and navigation in the mammalian brain. His work underscores the potential benefits of using new animal models in neuroscience.
After his talk Michael Yartsev was presented with an Eppendorf Research® plus pipette. His first reaction was “Wow, this is just what I wanted!” He was even more surprised to discover that his name had been engraved on the pipette.
More information at www.eppendorf.com/prize
New human 3D model system
Madeline Lancaster, Ph. D., Marie Curie Postdoctoral Research Fellow, winner of the Eppendorf Award for Young European Investigators 2014, also paid a visit to Eppendorf AG.
During her post-doctoral studies in the laboratory of Juergen Knoblich at the Institute of Molecular Biotech-nology of the Austrian Academy of Sciences, IMBA, Vienna, Austria, she succeeded in generating cerebral organoids (or “mini-brains”) from human pluripotent stem cells.
Using this 3D human model system opens up a whole new avenue in studying human neurological diseases.
More information at www.eppendorf.com/award
It has been a long-standing tradition to invite the winners of our Eppendorf research prizes to visit Eppendorf AG
Headquarters in Hamburg, Germany. Learning more about Eppendorf’s exciting company history and the great
versatility of the Eppendorf product world forms a major part of the visit. Additionally, the winners get in touch
with the people behind the products when presenting their research work to a broad audience of Eppendorf
employees.
Happy owner of a new pipette! Michael Yartsev will start his own lab in the summer of 2015 in the Department of Bioengineering at the University of California, Berkeley, CA, USA.
Madeline Lancaster visiting the Eppendorf Training Center. In 2015, she started her own lab at the MRC Laboratory of Molecular Biology in Cambridge, UK.
SERVICE · PRIZE COMPETITION
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The solution of the prize competition of BioNews No. 41 was “Cell Culture Consumables“. Ms Maria Varsami (General Hospital Papageorgiou, Thessaloniki, Greece) won the first prize.
Have fun with our new crossword!
How to find out the solution: Simply arrange the letters in the light gray boxes of the crossword in the correct order. Send us the solution until October 31, 2015.
You can either use the reply fax (p. 15), send us an e-mail to [email protected], or participate online at www.eppendorf.com/bn-service.
All correct answers will be considered for a prize. Winners will be notified in writing. Cash payment of the prize is not possi-ble. 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. 45.
ACROSS1 Stage in a process4 Elementary particles9 Term from the field of molecular
biology11 Person who flies an airplane13 Rapid eye movement (abbrev.)14 Take part in a game or sport17 Sultanate of the southeast Arabian
Peninsula18 European Union (abbrev.)20 Short form of Samuel22 Postal abbreviation of the US state
of Texas 23 ISO country code for Taiwan25 City in California (abbrev.)29 Skill or knowledge34 Piece of paper attached to an object
35 Fund provided to finance educational studies
36 Not two or more according to U238 Chemical symbol for platinum39 Process of gaining knowledge or skill43 Vessel used for holding flowers45 Opposite of rural46 To receive or take in the sense of
letters48 Refilled, recharged50 Makes Kong a city52 Italian composer53 Non-governmental organization
(abbrev.)54 Examinations, evaluations55 ISO country code for Austria
DOWN2 Interval, duration, period, era3 Energy and high spirits5 Common denominator of Associated
Press, alkaline phosphatase, and Asia Pacific
6 Short form of Brazil’s second largest city
7 Royal Dutch Airline (abbrev.)8 Cleansing agent9 Detailed plan for a scientific
experiment10 Internet slang for real life12 ISO country code for Tunisia15 Chemical symbol for arsenic16 University in New Haven, CT, USA19 Postal abbreviation of the US state of
Utah21 Mother of the Simpson family
(forename)24 Web seminar
26 Spanish aunt27 Written or printed message to
someone28 Settlement of accounts between banks30 Chemical symbol for xenon31 Scheme or method for attaining an
objective32 ISO country code for Turkey33 Single nucleotide polymorphism
(abbrev.)37 Electrically excitable cell40 Chemical symbol for rubidium41 Not chemically reactive42 Opposite of net43 Quo …?44 City in Yemen47 Compass point directly opposite to
west49 Spanish day51 Board game
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1st Prize:1 Multipette® M4* with 2 sets of Combitips advanced® of your choice *(U.S./CAN: Repeater® M4)
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Prize Competition
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E H I L T O T
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