AxoBits
Integrated Solutions for Cellular Neuroscience, Cell-B
Based Screening and Microarray Analysis z Vol. 41 z July 2004
GenePix 4000B Gets its Sea Legs
Dr. Alison Murray
Desert Research Institute
University of Nevada, Reno
As functional genomics moves from model
organisms to non-model organisms and
communities of organisms, new challenges
arise for researchers and their instruments.
Recently, an interdisciplinary research team,
headed by microbial ecologist, Dr. Craig
Cary, University of Delaware, embarked on
a 3½ week research cruise aboard the RV
Atlantis to the East Pacific Rise, 100 miles
off Costa Rica. Our mission, ‘Extreme 2003,
Depths of Discovery,’ brought together
several research groups including those led
by Dr. Eric Johnson of the University of
Oregon, and myself, Dr. Alison Murray of
the Desert Research Institute in Reno, NV.
The object of our mission was to delve into
environmental functional genomics by
detecting gene expression in biological
samples collected at deep-sea
hydrothermal vents over a mile beneath
the surface. At these sites, temperatures
fluctuate from four to over three hundred
degrees Celsius rapidly throughout the day,
and vent fluids are laden with heavy metals
(e.g. iron, manganese, and arsenic) and
high concentrations of sulfide. Despite
these extremes, Alvinella pompejana (the
Pompeii worm) and affiliated bacterial
episymbionts—a consortia of
predominately Epsilon proteobacteria—not
only tolerate the conditions, but flourish on
the vent chimneys. Little is known
regarding the specific details of the
symbiotic relationship between the worm
and its symbionts, how the worm
withstands the toxic environment, or how
the symbionts make their living. We hope
that gene expression surveys will help to
unravel the story of thermal adaptation,
tolerance, and function, as well as the
metabolic potential for these organisms.
Thus, our mission had three primary
objectives; sample collection, mRNA
processing, and testing the GenePix 4000B
scanner at sea. On a number of Alvin (the
submersible) dives, we collected the worms
in custom designed chambers that we
immediately flooded at depth with
RNAlater (Ambion, Inc.). This was only the
beginning of tricky science. Processing the
organisms to ensure the highest quality
mRNA is a technical feat, not to mention
the rest of the tasks involved in extracting,
labeling, and performing the hybridizations
(cont.)
themselves.
In This Issue
Product News
2
Special Pricing for PatchXpress
MultiClamp 3rd Party Control
GenePix 4200 Autoloader
GenePix Pro 6
Acuity 4
PatchXpress hERG News
Focus on Methods
4
The Mighty Model Cell
A Glimpse at Breast Cancer
Carbon-Fiber Microelectrodes
Tool Tips
6
DataXpress EC50 Script
Q&A
7
pCLAMP 9 Membrane Test
The Axoporator 800A,
single-cell electroporator,
is now shipping!
www.axoporator.com
The crew with Alvin and GenePix 4000B.
A MESSAGE FROM...
Alan Finkel,
Founder and CEO,
Axon Instruments
This will be the last issue of AxoBits to be
published under the Axon banner. We are
going through a rite of passage, being
acquired by Molecular Devices Corporation
and metamorphosing into a stronger,
combined company. Although our name
will change and we will be merged into a
bigger entity, it is my sincere hope and
indeed my intention that our efforts to
serve you will continue and that the
wonderful relationship that we have
enjoyed with you for twenty-one years will
be preserved.
I will be joining Molecular Devices as Chief
Technology Officer. Like a teenager leaving
cont. from Page 1...
Future experiments at sea will afford us the
opportunity to move from sample
collection to detection of gene expression
in as few as two days. This should allow us
to learn rapidly from our collection efforts,
and modify our sampling strategies when
needed. We plan to take a GenePix 4000B
on our next cruise, and hope to test gene
expression in the Pompeii worm, as well as
an environmental microbial genome array
to test hypotheses related to the metabolic
pathways utilized by the worm’s
community of episymbiotic bacteria.
For more information, visit
www.ocean.udel.edu/extreme2003/
home.html
Editor and Production
Simone Elletson
Marketing and
Public Relations
Simone Elletson
Debbie Quinn
While waiting for regulatory and
shareholder approval, we are continuing to
make and support our products, we are
developing exciting next generation
products in neurosciences, microarray
analysis and cell based screening, and we
are continuing to work with you, our
customers, to answer questions, solve
An Editor’s Reflections
Ships are challenging platforms to conduct
even the simplest of maneuvers required
by a land-lubbing functional genomicist.
Imagine trying to pipette 30 µl of a Cy-dye
labeled nucleic acid onto a 1x1 cm cover
slip in 10 ft seas in the dark! No less difficult
was the development of custom DNA
microarrays to study gene expression.
Amidst ship motions and vibrations, salty
sea air, and the occasional high seas the
GenePix 4000B worked perfectly, surviving
all these rigors for the entire voyage.
Editor-in-Chief
Al Walter, Ph.D.
home to go to college, I am a little scared
but full of eager anticipation. My job will
partly be a continuation of the traditional
Axon after-sales service; that is, to ensure
that the combined company efficiently
reaps maximum value from its purchase, by
assimilating and building on the best of the
Axon traditions. My other contribution will
be to the new product development effort
required to maintain the company as a
provider of leading edge, innovative
products.
Contributors
Jennifer Dent
Toni Figl, Ph.D.
Alan Finkel, Ph.D.
Dave Gallegos
Michael L. Heien, Ph.D.
Wilson Lew
Alison Murray, Ph.D.
Siobhan Pickett
James Rountree, Ph.D.
Damian Verdnik, Ph.D.
R. Mark Wightman, Ph.D.
Ward Yuhas, Ph.D.
2 · AxoBits 41 · www.axon.com
Although the AxoBits newsletter may
undergo a few changes following the
merger of Axon and Molecular Devices, its
focus−pointing the way to improved
methods and to new avenues of discovery−
will likely remain the same. We look
forward to the opportunity to draw from an
even larger pool of contributors, who like
those of the past will provide high-caliber
content in the form of application notes,
focus on methods and technical tips.
problems and generally respond as quickly
as always.
In many respects it is business as usual at
Axon, but like me, all of Axon’s employees
are feeling a mixture of sweet nostalgia
mixed with new hopes and expectations.
This will be my last message to you as CEO
of Axon Instruments, and in that capacity it
is my pleasure to thank you for your loyalty
and to state my strong hope that I will
continue to hear from you, or of you, as a
happy Molecular Devices customer.
Best wishes to you all,
Alan Finkel, Ph.D.
CEO, Axon Instruments
Special Academic Pricing
for the PatchXpress 7000A!
By now most of you have heard of the
revolutionary PatchXpress, Automated
Parallel Patch-Clamp system. The
PatchXpress is a 16-channel planar patch
clamp for increasing ion channel research
throughput. Academics can now enjoy a
special price to help them get this cuttingedge technology in their labs or shared-use
facility.
For a quote, please contact Anna Hastings,
at 510-675-6200 or [email protected].
For more information, visit
www.patchxpress.com
James Rae in his element.
Still there is a twinge of nostalgia on the
part of the current editor. I was fortunate to
inherit such a plum posting. Everyone who
contributed to the newsletter made my job
a labor of love. I am especially indebted to
the prolific duo of Drs. Jim Rae and Rick
Levis. Over the years they tackled a range
of patch pipette-related topics including:
perforated-patch recording, methods for
optimizing a system for ultra-low noise
recordings and, most recently, single-cell
electroporation. Apparently they draw from
an inexhaustible well of innovation and, in
my mind are true patch-pipette populists.
All the best,
Al Walter, Ph.D.
MultiClamp 3rd Party
Control Now Available
The MultiClamp 700A and 700B amplifiers
are each controlled by a software interface
(‘MultiClamp 700x Commander’). Although
this control interface is required to run the
amplifier, you can now develop your own
application to control MultiClamp 700x
Commander via a dynamic link library
(DLL). We hope this will allow those of you
with special needs and skills to use the
MultiClamp 700x in custom applications.
The DLL code and documentation is
available in MultiClamp 700A Commander
version 1.3 and 700B Commander version
2.1 (Windows versions only). These latest
versions are available on the MultiClamp
Updates page (see ‘Downloads’ at
www.axon.com).
PRODUCT NEWS
New! GenePix® Autoloader
4200AL Microarray Scanner
configured with up to four internally
installed laser excitation sources, either at
the time of purchase or as an upgrade at
any time afterwards. It also includes a
sixteen-position easy-access emission filter
wheel.
The Autoloader, like all GenePix scanners, is
compatible with 25x75 mm slide arrays
from Agilent, Amersham, Illumina,
Schleicher & Schuell, Combimatrix, and
most other sources. All GenePix scanner
models include GenePix Pro acquisition and
analysis software, and Acuity®, Axon’s fully
integrated microarray data warehouse,
informatics and visualization software.
For more information, visit:
www.genepixprofessional.com.
We are very pleased to announce the
launch of the GenePix Autoloader 4200AL.
This latest addition to our GenePix
microarray scanner product line fully
automates the entire process of microarray
scanning, image analysis and data
extraction.
The Autoloader automatically loads and
scans up to 36 standard microarray slides,
then analyzes the images and saves the
results—all without user intervention. Our
unique ‘never-let-go’ robot design ensures
zero slide breakage. Beginning with
microarrays in any slide format, the GenePix
autoloader uses line-by-line dynamic
autofocusing to yield unprecedented field
uniformity, even for warped slides.
Precision engineering and high-quality
components yield outstanding detection
limits, resolution, and repeatability. The
slides are scanned from a single easy-toload slide carrier, which includes a dust
cover for convenient stacking slide storage.
New spot-finding algorithms in GenePix Pro
6 allow highly reliable analysis of even the
most difficult images. With fully automated
batch scanning and image analysis you can
walk away from routine tasks and use your
time more productively.
The Autoloader is available as an upgrade
option from the GenePix Professional
4200A. Like the 4200A, the 4200AL can be
User-defined Columns
Custom columns are displayed in the
Results tab and can be used for Go To Web
queries.
Multiple Images
View side-by-side in GenePix Pro 6 by
opening multiple copies of GenePix Pro.
Acuity 4
GenePix Pro 6
GenePix Pro 6 is a landmark release in the
history of GenePix Pro. Our aim with
GenePix Pro 6 was to make microarray
analysis fully automatable, both for owners
of the new GenePix Autoloader 4200AL,
and to improve the productivity of our
existing customers.
Among the major new features in GenePix
Pro 6:
Automatable Spot Finding
We have revised and added to many of the
spot-finding algorithms that have not been
changed since GenePix Pro 3 was released
over 4 years ago. The result is a major
advance in our spot-finding technology, so
that spot finding is now completely
automatable.
Batch Analysis Tab
A new tab in GenePix Pro, together with the
revised spot finding, simplifies the batch
analysis of any number of microarrays. After
analysis, browse the results in GenePix Pro
with the new Browse Tool that successively
loads all the images and settings from the
batch.
Automatic PMT Adjustment
No more manual tweaking of PMT gain
before scanning an image! Our PMT
adjustment algorithm balances the signal
from all channels in your scanner and
maximizes their dynamic range, another
step towards full automation of microarray
scanning and analysis.
Morphological Background
Subtraction
The 36-slide magazine.
background that is higher than the
measured feature intensity. This solves the
problem of losing features from your
analysis that are highly expressed in one
channel but not expressed at all in the
other channel. Never again lose a feature
from your analysis because of high
background.
This new background subtraction method
ensures that you never have an estimate of
Acuity 4 is a major new release of Acuity,
Axon’s microarray informatics platform,
extending its functionality into several new
areas of data analysis and storage. Our aim
with Acuity 4 was to make it fully crossplatform compatible, both at the database
level by supporting the Oracle 9 database
platform, and at the microarray level by
fully supporting Affymetrix data analysis. In
addition, we have included many new
algorithms and visualizations to make your
data analysis even easier.
Among the many major new features in
Acuity 4:
Database Support: Oracle 9
Acuity now fully supports both Microsoft
SQL Server 2000 and Oracle 9, giving you a
choice between the two most powerful and
widely used database platforms available
today.
Data Warehousing
Store any file in the Acuity database,
attached to a microarray or dataset for easy
access. Import TIFF, JPG, GAL, GPS, PDF or
any other file relevant to your experiments
and keep all your data in the one place.
Never misplace an imported file again!
Affymetrix Import and Analysis
Axon has licensed the Affymetrix File SDK
so that Acuity can natively read and import
all Affymetrix file formats, such as CEL, CHP
and CDF. Acuity now includes Robust
Multichip Analysis (RMA), which is quickly
becoming the standard alternative to the
Affymetrix methods of normalization,
background subtraction and
summarization, so that you can get the
most from your Affymetrix data analysis
(cont.)
AxoBits 41 · www.axon.com · 3
FOCUS ON METHODS
Multiclass Classification and
Variable Selection
Build classifiers to find a minimal set of
genes that separates the different samples
in your experiment. This analysis is ideal, for
example, for cancer studies; in which you
are looking for genes that are diagnostic of
different types of cancer.
Chromosome View
Display expression levels from your
experiments directly on Acuity’s
chromosome maps, which can be
constructed for any genome for which
chromosome coordinates are available:
human, mouse, rat, yeast, fly, etc.
Advanced Graphing Abilities
Graph any quantity against any other
quantity in the Acuity interface—p-values,
correlation coefficients, data within
microarrays or across microarrays,
chromosome coordinates. Visualize the
data your way.
PatchXpress hERG News
In the late 1990’s, a number of drugs had to
be withdrawn from sale in the US when it
was determined that they caused
arrhythmias of the heart severe enough to
result in death. These drugs were found to
block hERG potassium ion channels .
Clearly, every potential drug should be
tested for hERG-blocking properties.
Unfortunately, there is no way to use a
drug’s structure to predict whether it will
block hERG. Therefore, hERG channel
testing remains the initial safety screening
method for candidate drugs.
Accurately testing hundreds of compounds
is a formidable challenge for individual
electrophysiologists working at manual
patch-clamp rigs. Such a process is slow,
tedious, boring, and wastes the talents of
highly trained and creative scientists. The
PatchXpress automates the screening
process providing high-quality data more
rapidly and cheaply. In cases where a
candidate drug causes hERG block, the
blocking concentration can be reliably
compared to those concentrations that
produce the candidate drug’s beneficial
effects on other targets.
Dr. Adrienne Dubin (J&J, San Diego, CA)
was the first to use the PatchXpress 7000A
with well-characterized hERG channel
blockers and compare the results to those
obtained with a conventional patch clamp
rig. Her findings are described in the hERG
101 article posted on our web site:
www.axon.com/cs_herg_screening.html.
4 · AxoBits 41 · www.axon.com
Optimization studies were recently
conducted by Quintiles and are detailed in
the Rapid ICE presentation:
www.axon.com/screeningnotes/
Quintiles_PatchXpress.pdf.
The Mighty Model Cell
Model cells are supplied with all of Axon’s
microelectrode amplifiers. Though simple
in design, model cells are incredibly
powerful tools for troubleshooting an
electrophysiology rig. Model cells are
designed to resemble a real recording
configuration; they are composed of high
quality resistors and capacitors that mimic
electrode resistances and passive
properties of cells. Because the
characteristics of the model cell do not
change, the results obtained with it must
always be reproducible. Thus, the model
cell is a perfect standard to test and
troubleshoot the rig. Some of the things
you can do:
• Check that the scaling factors are
correctly set in Clampex
(See Axon Knowledge Base Article
# 871)
• Narrow the search for the origin of a
drifting electrode potential
(See Axon Knowledge Base Article
# 281)
• Verify that the whole-cell
compensation circuitry is functioning
properly
(See detailed tutorials of the Theory
and Operation manuals)
If the passive properties of the cells from
which you record differ greatly from those
of the Axon model cell, then you may want
to construct a custom model cell. The
Membrane Test feature in Clampex is one
way to determine the values for the cell
and a patch electrode. (See Axon
Knowledge Base Articles # 649 – 652.)
Although you need not be able to read
schematics (provided in the Theory and
Operation Manuals), you must have a
rudimentary understanding of how the
parts function. You can access the innards
of an Axon model cell and trace the
connections that you will make with the
components that you choose for your
custom model cell. Typically the
components purchased at a nearby
electronics store will not be of the same
high quality as those used by Axon. Even
so, you should be able to predict a signal’s
scaling and time course based on the value
of the components selected. Naturally, it is
best to verify the performance of the
custom model cell on an amplifier that has
been found to be functioning properly with
an Axon model cell.
None of the model cells sold by Axon have
an internal power source and, therefore,
need no maintenance. In rare instances
one might wish to emulate either a
junction potential or resting membrane
potential. The details for construction are
described in Article # 784 in the Axon
Knowledge Base.
Keep Your Model Cell Handy At All Times!
When a problem arises, you can count on it
to point the way to the guilty or innocent
element(s) of your rig.
For a very detailed discussion of model
cells, please see:
http://fisio.dipbsf.uninsubria.it/scuola/patch.pdf
A Glimpse at Breast Cancer
Gene Profiling
Jennifer Dent
Combimatrix
Breast cancer researchers have used
microarrays to define subclasses of the
disease, monitor progression, and guide
treatment based on genetic expression
profiles. It is essential that custom, highquality microarrays be produced in a matter
of days to keep pace with evolving needs
and speed the discovery process for all
types of genetic research. CombiMatrix’s
CustomArray™ 902 is such an array. Gene
targets can be submitted by GenBank
accession number, or by sequence, through
a web-interfaced software suite that
includes algorithm-based probe design.
CustomArrays are produced using a
semiconductor-based, electrochemicallycontrolled in situ synthesis process that
(cont.)
Figure 1. Custom array slide visualized in GenePix Pro
FOCUS ON METHODS
allows researchers to specify any set of
genes from any organism. The array is
embedded in a black alumina base that
conforms to the standard 1" x 3" slide
format, making it fully compatible with
GenePix scanners. Standard methods for
sample preparation and hybridization are
utilized when working with CustomArrays.
Two-color comparative hybridizations of
breast cancer genetic markers compiled
from recent literature and public databases
were performed using CustomArrays.
MCF7 (breast adenocarcinoma) cell line and
mammary gland poly-A+ RNA were
processed through a modified Eberwine
amplification procedure, resulting in Cy3
and Cy5 incorporated cRNA. Equal
amounts of labeled MCF7 and mammary
gland cRNA were combined and
fragmented, followed by overnight
hybridization to the CustomArrays. Images
were collected using the GenePix 4000B
microarray scanner (Fig. 1), and then
analyzed using GenePix Pro and Acuity.
Differential expression was easily visualized
on the two-color image, and ratio data
from Acuity showed strong correlation to
published gene expression data.
To learn more about CustomArray™902,
visit www.customarray.com.
Carbon-Fiber
Microelectrodes:
Fabrication and Treatments
Michael L. Heien and R. Mark Wightman
University of North Carolina at Chapel Hill
Since the early 1980s, carbon-fiber
microelectrodes (CFMs) have been widely
used to measure concentration changes of
easily oxidized molecules in biological
preparations. CFMs can be used with
constant applied potential (amperometry)
to achieve high temporal resolution
recordings of transient secretion of
neurotransmitters.1 Alternatively, CFMs can
be used with fast-scan cyclic voltammetry,
which has lower temporal resolution but
allows identification of different molecular
species.1 For instance, exocytotic release of
dopamine, norepinephrine, serotonin, and
histamine have been measured from single
cells in culture. Individual secretory events
can be clearly resolved, and the time course
of secretion easily followed.2 Measurements
have also been made in intact preparations
such as a brain slice or anesthetized animal,
where release and subsequent cellular
uptake of dopamine and serotonin have
been characterized.3
Microelectrodes well-suited for biological
measurements must meet the following
criteria:
Visit the Axon Booth at:
Advanced Microarray Strategies for
Biopharmaceuticals
Boston, MA, June 6-9, 2004
4th Forum of European Neuroscience
Lisbon, Portugal, July 10-14, 2004
SBS 10th Anniversary Conference and Exhibition
Orlando, FL, September 11-15, 2004
Chips to Hits
Boston, MA, September 21-23, 2004
Ion Channels in Drug Discovery & Development
Philadelphia, PA, October 4-5, 2004
Assays & Cellular Targets
San Diego, CA, October 17-21, 2004
Society for Neuroscience
San Diego, CA, October 23-27, 2004
American Society of Human Genetics
Toronto, Canada, October 26-30, 2004
American Heart Association
Scientific Sessions 2004
New Orleans, LA, November 7-10, 2004
The American Society for Cell Biology
Washington, D.C., December 4-8, 2004
• The electrode and its accompanying
insulating material must have small
dimensions to operate in the
compact extracellular space. Currents
generated at the electrode must be
small (< 1 µA), so as not to disturb
the surrounding tissue.
• The electrode time constant must be
kept low to enhance temporal
resolution, making high-speed
applications possible.
The workhorse that satisfies the above
criteria is the glass-encased CFM. This note
provides an overview of how CFMs are
manufactured.
Construction of Electrodes
Over the years, we have developed a
straightforward method for production of
electrodes.4 It should be noted that several
commercial sources of carbon-fiber
microelectrodes now exist and represent an
alternative to the do-it-yourself approach.
Both disk-shaped and cylindrical electrodes
can be made with carbon fibers. Fibers in
the order of 3 to 30 µm in radius are very
delicate and have a relatively high
resistivity. Resistance is kept low by using a
short section of the carbon fiber for
construction.
With patience and practice, even a novice
electrode maker will soon find handling
these small fibers effortless. A single carbon
fiber (Goodfellow Corporation, Berwyn, PA)
is visualized and a pair of watchmaker’s
forceps with the tips encased in heatshrink
tubing is used to handle the fibers and
remove any debris. The carbon fiber is then
aspirated into borosilicate capillary glass
(0.6 mm ID, 1.2 mm OD, A-M Systems,
Everett, WA) so that it protrudes from both
ends of the capillary. The carbon fiber-filled
capillary is then placed in any modern
micropipette puller, and the trial and error
process for finding the optimal settings
begins. The pulled glass must form a fluidtight seal around the carbon fiber. A light
microscope will reveal visible cracks or
irregularities in the glass seal.
If the electrode passes the first production
step, it is ready to be processed to the
desired geometry. A cylindrical geometry is
easily produced by cutting the exposed
carbon fiber to the desired length, typically
50 to 150 µm. This is accomplished with a
sharp scalpel under a light microscope
(100× magnification). A cylindrical CFM is
depicted in Figure 1. The electroactive area
of this electrode protrudes approximately
100 µm from the glass seal. Because the
signal measured is directly proportional to
the electroactive area, cylindrical electrodes
are used in experiments where a larger
electroactive area is desired, such as a brain
slice or a whole animal preparation.
Figure 1. A cylindrical CFM.
For experiments where more spatial
specificity is desired, disk electrodes are
used. To fabricate this type of electrode, the
tapered portion of the pulled capillary is
trimmed with a scalpel to a diameter
slightly larger than the carbon fiber itself.
The tips are then dipped in epoxy to obtain
a seal between the glass and the carbon
fiber (Epon 828 with 14% m-phenylenediamine by weight, Miller-Stephenson
Chemical Co., Danbury, CT). The epoxy
must be heated to > 80° C to decrease the
viscosity, so that capillary action draws the
epoxy into the gap between the carbon
AxoBits 41 · www.axon.com · 5
TOOL TIP
fiber and the glass. The epoxy is then cured
(100 °C for 12 h, 150 °C for 2 days). The tip
of the microelectrode is lowered onto a
beveler (BV-10 Micropipette Beveler, Sutter
Instrument, Novato, CA) and polished at an
angle to produce an oval cross section of
the carbon fiber. A disk microelectrode can
be seen in Figure 2.
change the surface properties (as
demonstrated by scanning electron
microscopy11) and increases surface
oxides.12 This increases the sensitivity of the
electrode to analytes such as dopamine,
but causes a slower electrode response.
CFM capacitance should be minimized to
make low-noise measurements. The
capacitance has two components, the
capacitance across the glass and the larger
contribution from the electrical double
layer at the electrode tip. These two
components are kept to minimum in two
ways:
• By producing a well-insulated
electrode, solution cannot creep in
between the carbon fiber and
surrounding glass pipette.
• By keeping the electrode surface
smooth.
Figure 2. A disk microelectrode.
The electrode is backfilled with electrolyte
(4 M potassium acetate, 150 mM potassium
chloride) and fitted with a wire that
connects to the input of the
electrochemistry equipment. An electrical
contact can also be made by using colloidal
graphite, silver paint, or mercury.
Electrode Pretreatments
Although one can use freshly
manufactured electrodes, there are several
pretreatments that can dramatically alter
the surface properties of the carbon fiber.
These will create CFMs that have their own
unique measurement characteristics.5 The
most common treatment is the use of
Nafion®, a perfluorosulfonated ion
exchange polymer, which acts to exclude
anions from the electrode surface while
concentrating cations.6 This enhances
selectivity, and protects the electrode
surface from fouling.
In summary, CFMs are wonderful tools for
measuring concentration changes of a wide
variety of easily oxidized biomolecules.
These electrodes can be manufactured to
meet the constraints of space and response
bandwidth and their specificity can be
optimally tuned by using proper
methodology combined with selective
well-characterized treatments.
Many electrochemical treatments work to
enhance the adsorption and electron
transfer kinetics of the electrode surface. In
fact, it has been remarked that there are as
many electrochemical treatments as there
are labs using carbon electrodes. Most of
these involve applying a positive potential
to the electrode to obtain the desired
results.8-10 Such treatments dramatically
6 · AxoBits 41 · www.axon.com
The ‘Voltage-Gated EC50 (no rundown)’
script in DataXpress calculates doseresponse relationships and effective
concentration (EC) values for sets of test
compounds. This article provides some
background information to help set up the
script.
The script measures the peak current in
every sweep for each cell procedure in the
dataset (a ‘cell procedure’ is the application
of the defined experimental procedure to
one cell). It plots the measurements for
each cell procedure against time (Figure 1).
The measurements could come from one
continuous recording run throughout the
procedure, or numbers of separate
recordings run sequentially—the script
treats both scenarios the same. The relative
effect of each dose is used to generate a
dose-response graph, which is fit with a Hill
curve. The Hill equation yields the effective
concentration for a particular percentage of
the maximal effect—typically 50%, hence
‘EC50’ in the script name.
References
1.
2.
3.
4.
If a CFM is soaked for at least 10 min in
isopropanol purified with Norit A activated
carbon (ICN, Costa Mesa, CA), the sensitivity
can be improved by more than threefold.7
Presumably this cleans the surface and
changes the surface groups on the
electrode. This cleaning step is especially
important in cases where the
microelectrode’s surface cannot be
polished due to geometry.
DataXpress EC50 Script
5.
6.
7.
8.
9.
10.
11.
12.
Cahill, P.S., Walker, Q.D., Finnegan, J.M., Mickelson, G.E.,
Travis, E.R., Wightman, R.M. Microelectrodes for the
measurement of catecholamines in biological systems,
Anal. Chem., 68:3180-3186, 1996.
Wightman, R.M., Jankowski, J.A., Kennedy, R.T., Kawagoe,
K.T., Schroeder, T.J., Leszczyszyn, D.J., Near, J.A., Diliberto,
E.J., Jr., Viveros, O.H. Temporally resolved catecholamine
spikes correspond to single vesicle release from individual
chromaffin cells, Proc. Natl. Acad. Sci. USA. 88:1075410758, 1991.
Millar, J., Stamford, J.A., Kruk, Z.L., Wightman, R.M.
Electrochemical, pharmacological and
electrophysiological evidence of rapid dopamine release
and removal in the rat caudate nucleus following electrical
stimulation of the median forebrain bundle, Eur. J.
Pharmacol. 109:341-348, 1985.
Kawagoe, K.T., Zimmerman, J.B., Wightman, R.M. Principles
of voltammetry and microelectrode surface states, J.
Neurosci. Meth. 48:225-240, 1993.
McCreery, R.L., Cline, K.K. Carbon Electrodes, Laboratory
Techniques in Electroanalytical Chemistry , 2nd ed. P.T.
Kissinger, W.R. Heineman. Eds. New York: Marcel Dekker,
Inc, 1996.
Gerhardt, G.A., Oke, A.F., Nagy, G., Moghaddam, B., Adams,
R.N. Nafion-coated electrodes with high selectivity for CNS
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Bath, B.D., Michael, D.J., Trafton, B.J., Joseph, J.D., Runnels,
P.L., Wightman, R. M. Subsecond adsorption and
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Anal. Chem. 72:5994-6002, 2000.
Gonon, F., Buda, M., Cespuglio, R., Jouvet, M., Pujol, J.F. In
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improved sensitivity to dopamine with extended
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Figure 1. Peak measurements over time for a single cell,
showing compound additions. In this example 6 trials
were run, as evidenced by the gaps in the data.
A minimum of four points is required to
apply the Hill fit to dose-response data.
Two extra points, at the top and bottom of
the data range, can be added in the script,
so data with as few as two concentration
points can be fit. The script will also run
with data that have a different compound
added after the test compound, for
example, a dose that completely blocks all
channel current. Such a ‘hammer dose’ is
not at this time used in the calculation of
current inhibition, but this feature will be
incorporated soon.
To prepare a dataset, view the trials
returned from your original query and
remove bad trials by selecting them in the
trial list and then hitting Control + Delete.
Use the Trial Editor to remove anomalous
sweeps and spikes.
When the trials are ready for analysis, set
cursors 3 and 4 to the region of the sweep
where you want to measure the peak, and
cursors 1 and 2 to a baseline region (unless
you intend to use the absolute peak
measurements option in the script). Then
Q&A
go to the Script tab and open ‘VoltageGated EC50 (no rundown)’. By opening the
script after the trial has been opened in the
Trials tab, the script’s ‘Use signal’ list box
automatically shows the number of signals
that the trial contains, starting with ‘0’.
PatchXpress users who enabled P/N leak
subtraction will generally select the second,
leak-corrected signal, ‘1’.
When the script runs, it takes
measurements from the final five control
data points prior to drug addition (Figure
4). The average of these peaks becomes the
maximal response against which inhibition
values for each test compound
concentration is calculated.
Question:
In pCLAMP 9, I would like to be able to
follow the Membrane Test parameters (Ra,
Rm, and Cm) graphically during my
experiment. It would be helpful if the
graphical information were stored so that I
could return to it later in the experiment
and analyze the trend.
Answer:
The ‘Control is compound addition number’
field determines the peaks used to set the
reference value for the compound effect for
each cell. To find the correct compound
addition number, go to the Cell Procedures
tab, highlight a cell procedure from the list,
right click, and select ‘Compound Additions’
to bring up the Compound Additions
dialog for the highlighted procedure
(Figure 2).
Figure 2. The Compound Additions dialog shows all
fluidics steps in the cell procedure. The ‘control’ step is
the one prior to the first addition of test compound.
The dialog shows all the fluidics events—
washouts and compound additions—for
the selected cell procedure, in the order
they occurred. Each row in this list is
considered, for the purposes of the script, a
compound addition, and the ‘control’
compound addition is the one prior to
application of the first concentration of test
compound. For example, in Figure 2, the
first concentration of test compound
(CmpndA in 0.004% DMSO) is in row 3, i.e.,
compound addition number 3, so the
‘control’ compound addition is number 2.
It could be that the only ‘compound
addition’ prior to application of the test
compound is a washout—in this case then
the washout is taken to be the control
compound addition, number 1. Note that
the script requires that there is at least one
fluidics event before test compound is
applied.
The rule is the same if you have double
dose compound additions enabled. This is
the case in Figure 3. In this example, the
first addition of test compound is addition
number 4, making addition number 3 the
control for the purposes of the script.
Figure 3. Compound additions in a cell procedure with
double dose compound applications configured.
You can set up Clampex 9 to show a
graphical output of the Membrane
Parameters that will be stored throughout
the acquisition session.
Figure 4. The last n peak measurements following
addition of control and before the first addition of test
compound are used to set the maximal response value.
The number of peaks to average is set in
the ‘Peaks to average for control’ field. For
example, if you enter ‘5’ here, the last five
peak measurements prior to application of
test compound are averaged (again, Fig. 4).
A different number of peak values can be
used to determine the average current
magnitude following additions of test
compound.
As mentioned above, you can opt to
include zero-response and/or 100%
response points in your dose-response
graph. A zero-response point is added two
log values below the lowest concentration
tested, and the 100% response point two
log values above the highest concentration
tested.
Start the Membrane Test by pressing the
appropriate button (the button looks like
an RC circuit with a 0 or 1 inside it). This
action should open the Online Statistics
window automatically (in addition to the
Membrane Test window). By default, Rm
and Cm are graphically tracked. To add or
delete statistics for the Membrane Test,
click on the Options button within the
Membrane Test window. In the Results
Display section, check the parameters that
you want displayed in the Statistics window
(you will need to close and reopen the
Membrane Test).
Finally, set the effective concentration value
to be calculated, for example the EC50, and
hit the Run button. A dose-response graph
reporting the selected EC value is
generated for each cell procedure (Figure
5).
Figure 5. Final dose-response graph for one cell
procedure, fit with Hill and EC value calculated.
In the analysis summary on the Scripting
tab, result values for each test compound in
the dataset are reported. If you select
‘Graph and Data’ or ‘Data Only’ display, you
can copy the numerical results for
individual cell procedures to the clipboard
for further analysis in a spreadsheet
application. Note that you must click in the
data region of the Results tab before
copying.
Unless cleared by pressing the Clear
Statistics button, the Membrane Test data
will accumulate in the Online Statistics
window. By changing the x-axis extent
using the zooming triangles to the left of
the x-axis, you can enlarge or reduce the
time base, thereby focusing on the trend of
the Membrane Test statistic that is relevant
for your experiment.
Questions?
The Axon Knowledge Base
has the answers!
www.support.axon.com
AxoBits 41 · www.axon.com · 7
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