Assay and Drug Development Technologies, Vol.5 Number 4, 2007

ASSAY and Drug Development Technologies
Volume 5, Number 4, 2007
© Mary Ann Liebert, Inc.
DOI: 10.1089/adt.2007.9990
An Interview with Lars J. Brandén, Ph.D.
Associate Director for Chemical Genetics, Judith P. Sulzberger
Genome Center, Columbia University, New York
Lars J. Brandén, Ph.D., is Associate Director for Chemical Genetics at
the Judith P. Sulzberger Genome Center, Columbia University, in New
York City, as well as Director of Automation for the Columbia Molecular
Library Screening Center Network, in the Department of Biophysics and
Cell Biology. Dr. Brandén received his undergraduate degree in
molecular genetics from the University of Lund, Sweden, and completed
his doctoral studies in cell and molecular biology at the Karolinska
Institutet, Stockholm, Sweden, under the guidance of Professor C.I.
Edvard Smith. He pursued a postdoctoral research fellowship in the
Laboratory of Cellular Biochemistry & Biophysics at the Sloan-Kettering
Institute, Memorial Sloan-Kettering Cancer Center, New York City, under
the direction of James E. Rothman, Ph.D.
465
Dr. Brandén, how does the work underway in your labo-
ratory at the Judith P. Sulzberger Genome Center fit in with
the National Institutes of Health’s (NIH’s) Molecular Li-
brary Screening Center Network (MLSCN)?
The whole setup we have here was initiated because
we received the MLSCN grant. The 6,000 square feet of
space we received was intended to be refurbished and used
to support the NIH molecular library screening program
and to establish a network lab. The work I do is almost
exclusively for the MLSCN. We have another grant, a
philanthropic donation that supports our embryonic stem
cell research; it is not related to the MLSCN activity.
The lab includes an automation suite where the screen-
ing takes place, divided between three robotic platforms
that are clones of each other. All the automation equipment
is enclosed in Biosafety Level 2 enclosures, and the room
itself has been designed to be at Biosafety Level 2ϩ. The
space has welded floor seams, special paint on the walls
that can withstand harsh detergents, ceiling tiles that are
dust resistant, and magnetic locks on the automation room
so we can control access. The lab also has two cell culture
rooms; one is dedicated to R&D cell culture, and the other
for high-throughput production of cells. The room for high-
throughput cell production also houses embryonic stem cell
culture. My intention was to have as few people in that
room as possible, so I have separated it from the space used
for everyday types of R&D cell culture applications, to
which a lot of people need in-and-out type of access daily.
The high-throughput cell production room features an au-
tomatic cell culture system we developed here. It was de-
signed with an airlock, in which people switch gowns. It
has in situ autoclavable incubators that can be cycled up to
about 120°C to avoid contamination risk. Their interiors
are copper, which minimizes growth inside the incubators.
The freezer room houses the cell bank. When we ex-
pand the cells we can freeze them down at high density
in a controlled-rate freezer to maximize cell viability. We
bank our cells in liquid nitrogen. We also have a room
with a colony picker for library replication, but that is
not intended for use by the MLSCN project. In that room
we also have the gas supply for the automation suite—
carbon dioxide and nitrogen tanks—so we do not need
to go into the automation room to switch gases.
The wet lab is a standard wet lab that can house up to
18 people. In the equipment niche we have a Tecan
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MiniPrep, shakers, and incubators. The floor space needed
for supplies to support the R&D work and for high-
throughput production is removed from the general lab to
maximize use of the space. The office suite was designed
to be like a hub, where everyone can gather, to promote
interaction between the HTS and the biology core, for ex-
ample. My intent was for the core people to have easy ac-
cess to each other, so the office suite is designed as an open
structure with a few dedicated offices. The chemistry group
works across the street from us. We are integrating them
quite heavily by bringing them over on a regular basis—
once or twice a day—and providing open office space in
which they can easily interact with the rest of the group.
Our informatics core is in Florida. We have a dedicated
T1 line, so we have no problem with dedicated data trans-
fer. We can transfer as much as 50 terabytes of data per
year. The lab team has traveled to Florida on retreats, and
we have had very good feedback from those experiences;
they engender a sense of belonging. We have dedicated
informatics hardware and personnel in Florida—they work
exclusively with us—and we benefit from the experience
and network located there. They are the best of the best!
I am trying to integrate the findings and reagents we
produce from my other projects into the MLSCN proj-
ect. For example, our embryonic stem cell project in-
volves cloning all the human genes and tagging them with
green fluorescent protein (GFP). We have about 17,400
genes now, and I am waiting for another 1,400 to be
added to our collection. Suitable GFP fusions can then
be transferred into an assay, and we can capitalize on that
project to introduce new assays into the network. We try
to synergize as much as possible.
What part does chemical genetics play in this overall ef-
fort, and how are you applying HTS and robotics to study
gene function and establish links between gene expres-
sion and human disease?
We are gearing up to use HTS and robotics to study
gene function. We need a gene expression library avail-
able before we can start doing gene screens. The embry-
onic stem cell project I am running is establishing the ge-
nomic reagents needed to do these gene screens. The NIH
allows us to use the equipment and personnel from the
MLSCN project—when there is time left over—to pur-
sue our other work, and we will take the genomic reagents
we produce and screen them against cell-based assays in
which we have identified compound hits. We can look at
which of the genes we have overexpressed or knocked
down that are able to shift the 50% inhibitory concen-
tration of the drug, indicating that the gene plays an im-
portant role in the pathway in which this compound in-
teracts. There will be a lot of generic hits—hits affecting
multiple pathways, for example, drugs that disrupt vesi-
cle transport, especially in an assay such as nuclear fac-
tor ␬B (NF␬B), where you have recirculation of the re-
ceptor. If you disturb that by disrupting vesicle transport
it can mimic a hit, because you will be preventing translo-
cation of the receptor back to the cell surface. You can
no longer stimulate the cells and it will look as though
you are blocking the pathway.
You can generate the same kinds of generic hits when
it comes to genes—genes that affect a lot of different
pathways without necessarily targeting a particular path-
way. An example would be some of the more generic ki-
nases. When a particular gene is overexpressed you might
see some functions appear that would not be present at a
lower expression level. Similarly, with some compounds
you might see more specific effects when they are pres-
ent at low levels, whereas at higher concentrations a com-
pound may exhibit a greater breadth of functions but
lower affinity for a particular target.
Gene screens will require high content analysis; both
time course and concentration studies might be necessary
so you can see when certain effects appear and disappear.
Our goal is to produce three sets of genomic reagents to
enable us to convolute linked pathways: a cDNA over-
expression library with corresponding short hairpin RNA
(shRNA) constructs and the corresponding cDNA fused
to GFP.
With regard to establishing links between gene ex-
pression and human disease, if you have an assay that
describes a disease state, you can overexpress gene by
gene and look for a shift in the disease model. You can
do the same thing with shRNA to knock down gene by
gene. You can then establish which genes are involved
in a disease platform.
What screening platform are you using, and how was it
customized for your lab? How are compound libraries
interfaced to the system?
We are using the Tecan screening platforms (Fig. 1).
They are not really customized, but we have adapted them
extensively. For example, we have added an extra lid
chute that takes care of the lid problem. Once you seal a
plate with sealing tape you cannot re-lid it, so the lid has
to go somewhere. When the robot stacks the plates in the
platform a chute going through an opening directs the
lids into a trashcan.
We also have a custom-made hand-off station. Two ro-
botic arms are located on either side of the platform. The
left arm cannot reach all the way to the right side, and vice
versa, because there is a liquid handling arm situated be-
tween them. So the left arm moves a plate as far as it can
to a hand-off position and stops there. The hand-off station
then transfers the plate to the robotic arm on the right side.
We have not completely automated the compound li-
brary handling. However, when we load the compound
library into the automation platform the plates are bar-
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coded, so we can trace the location of each compound
and cherry-pick from a particular well. But the actual
handling of the compounds, such as loading into the
carousels, is manual. Automating a complete freezer sys-
tem, or even a room temperature storage unit, and au-
tomating cherry-picking and loading into the automated
platform would be very costly. With that same amount
of money we can hire about three more full-time equiv-
alents who can serve other functions as well. The trans-
fer process only involves accessing the compounds be-
fore a run and returning them to storage after a run. When
you start screening large compound libraries, then it is
helpful to have the automation in place; we are investi-
gating how we will meet that need when it arises.
What is the follow-up procedure you use to classify ac-
tives from your screens?
Once we have identified an active compound in a pri-
mary screen we redo the screen on the identified com-
pound, and once we have verified that it is an active, we
then run a titration curve. We are in the process of de-
ciding whether we should run a titration curve right away
on a hit, or whether we should wait for the verification
that it is a real hit. About 80% of hits are false-positives.
Do you have a different procedure for handling libraries
of shRNAs or other biomolecules?
Compound libraries can remain at room temperature
for about 3 or 4 months according to the Lipinski guide-
lines. I would not let a short interfering RNA (siRNA)
or shRNA plasmid library sit out at room temperature for
that period of time. We have to aliquot those more strin-
gently. We are more likely to go through three or four
cycles with siRNA, shRNA, plasmids, or cDNA overex-
pression plasmids, for example. If we opt to use adeno-
virus, we can keep them at ϩ4°C for a couple of weeks
without losing the titer. So for adenovirus-based libraries
you can aliquot out what you need for 2–3 weeks.
Lentiviruses are more problematic. You can freeze and
thaw them, but you do lose titers, depending on the pro-
cedures you use. With standard freeze/thaw procedures
you can lose up to 70% of your titer. If you use a mod-
ified protocol you may be able to maintain up to 80% of
your titer through three or four freeze/thaw cycles, but
then it will drop off. Lentiviruses are usually very un-
stable at 37°C—they have about an 8-h half-life. You
need to have a good understanding of lentiviruses to work
with them efficiently.
What technology do you have in place for cellular imag-
ing, and what advantages and limitations does it have?
We use the IN Cell Analyzer 3000 from GE Health-
care. We have about 15 different algorithms for studying
various events within cells, including object intensity and
nuclear translocation algorithms.
The main limitation is true confocality with high res-
olution in the z orientation. We are working with rela-
tively flat cells—they are at most a couple of microns
thick—but it would be beneficial to be able to work on
other types of cells from a confocal standpoint. Several
systems on the market can achieve that with high through-
Profile 467
FIG. 1. Columbia screening center automation platform. The Columbia screening center automation consists of three identical
TECAN platforms housed in BLS-2 enclosures. Each system has been designed for maximum flexibility and can process a wide
assortment of assay types as well as performing various types of cell culture and virus production (adenovirus primarily but also
lentivirus). Each platform has an integrated plate reader to enable seamless processing of low-content cell-based assays or enzy-
matic assays.

put, such as the Opera™ system from Evotec Technolo-
gies, the BD Pathway™ 435 system from Becton
Dickinson, and the UltraVIEW™ LCI system from
PerkinElmer. I wouldn’t single out any one system—they
are all based on a spinning disk system and can give very
good four-dimensional resolution.
We also have plate readers, which are quite useful for
lower content analysis. They can do virtually any type of
assay except Flash reading; they can do fluorescence res-
onance energy transfer, fluorescence, luminescence, and
absorbance.
How are you using imaging to do assay characterization
of your candidate compounds from HTS?
We have a large set of assays developed in-house,
about 130 of them. We select a cross-section of those de-
termined to be most relevant for a particular study and
run the candidate compounds on them. These assays can
help determine, for example, how specific the compounds
are; if a compound activates only a limited number of as-
says, you can then determine whether those assays are
related to each other. For instance, if you were to get a
hit in the NF␬B pathway, and you could see that it was
propagated through the vascular cell adhesion molecule
(VCAM) and E-selectin pathways, and you knew that
these pathways were on the same “line”—first NF␬B,
then E-selectin, then VCAM—then you would have a
very interesting finding. If you had a “hit” in the VCAM
pathway that did not activate the NF␬B or E-selectin
pathways, then you might have a very selective hit. You
can do a lot of deconvolution by looking at the results of
specific assays and determining where your hits pop up
in them. You can also learn something about when the
hits manifest in the different pathways. For example, if
you have a block in an upstream pathway that is feeding
a secondary assay downstream, you can say something
about when it should it be turned on.
Our aim, with regard to the use of cell-based assays, is
to allocate them extensively and to annotate them carefully.
We are trying to characterize the assays we have in-house
on one particular cell type, human umbilical vein embry-
onic cell (HUVEC) primary endothelial cells. If we were
to switch from cell type to cell type, we would get lost. By
focusing extensively on one cell type for the characteriza-
tion studies, you can then switch to a different cell type and
see what differs. Then you can start understanding why dif-
ferent drugs may have different potencies, for example, or
why their side effects are different.
What are important considerations when setting up a
large-scale tissue culture lab for HTS?
The most important factor is to minimize the risk of
contamination, to the nth degree. Our large-scale tissue
culture setup is an integrated system. It is based on spin-
ner bottles and microcarriers (Fig. 2). When we build a
system, we fuse together all the associated tubes with a
sterile welder, and we use a 0.2-␮m filter for gas ex-
change, which is the only opening in the system. The en-
tire system is autoclavable. We then connect the system
to the media supply using a sterile welder to seal the bot-
tles to the tubing. There is never an opening to the ex-
ternal world, not even inside laminar hosing. The only
time that happens is when you put the sterile cap onto
the media bottles, which is done inside of a hood. We in-
oculate via a septum.
To expand the cells, before we inoculate them into the
high-throughput production unit, we use Corning® Cell-
STACK® culture chambers and put them in incubators
that cycle up to high temperatures, up to 120°C, as de-
scribed previously. The entrance to the high-throughput
cell production lab has a small airlock; the door never
opens straight into the cell culture room.
How does one keep cell response stable over the dura-
tion of a multiday screen?
First, you have to have done your homework on the
bench regarding your assay. If the assay demands that the
cellular response should be stable over the duration of a
multiday screen, and that is not feasible with the proto-
col as written, then that cell line cannot be used in a mul-
tiday assay. If you have an assay that is supposed to be
reactive on the first day and you have a plate that comes
into the process a day and a half later, for example, then
you must determine the viability and utility of the cells.
We have studied how long we can keep the cells in plates
and maintain the same output and response. That time
limitation sets the limit for the batch size. The batch size
depends completely on the biology.
One solution is to use a Ϫ80°C carrier, or “hotel,” on
the platform, which can house cryogenically preserved
cells together with, for example, cytokines. We have a
trough in the incubator filled with regular media (with-
out cytokines). We can then thaw the cells and the cyto-
kines and spike the media on demand. Even if the in-
tegrity of the cells is such that we cannot use them for
more than 24 h, we can feed out additional cells on de-
mand using this in-house system. We can maintain com-
plete homogeneity from the first plate to the last in a large
batch using this approach.
What are the best viability criteria and detection meth-
ods to decide cell health for good assay response?
We always run a validation screen on the cells we plan
to use. We determine if they respond as expected to con-
trol compounds. If, on the validation run, we see a loss
of cell number or reduced activity of the cells, then we
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know that something is wrong, and we need to evaluate
the problem. Something as simple as cell count will tell
us if there is a problem with cell viability, and there are
a variety of cell viability and toxicity assays available.
We feel it is best to run the assay with a few control com-
pounds and see if we get the expected results.
For engineered lines, are there specific cell types or
parental lines that reliably give a good response?
HUVECs are primary cells, not a cell line, but that is
the specific cell type that gives us the best response. They
contain many of the components of the signaling path-
ways in which we are interested. These endothelial cu-
bic cells are very useful—they line all the blood vessels
and are the first line of response in inflammation. They
have many receptors and are readily stimulated. They also
have a good representation of the majority of linked path-
ways.
Do you use transiently transfected cells in HTS? If so,
how do you accomplish this?
No, we do not. If we get an assay that demands tran-
siently transfected cells, then we take a close look at the
protocol to see if we could change to a cell line that is
more easily transfected. Personally, when it comes to
transfection of cells, I prefer to use inexpensive trans-
fection reagents rather than one of the more costly alter-
natives. If we would use a transiently transfected cell line
for HTS, we would use something that could be done
very cheaply in-house.
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FIG. 2. Columbia screening center high-throughput cell culture system. The Columbia screening center high-throughput cell
culture system is designed around three major components: (1) the spinner flask culturing system from Corning (schematics shown
on upper left, spinner flasks shown on lower right); (2) the Delta V controller from Broadley-James; and (3) the BioNet soft-
ware running the system (both upper middle). (Lower left) Top view of growth and harvest flasks. By integrating these systems
we have an in-house–developed high-throughput cell culture system that can produce in the range of 4 ϫ 109 cells per batch. This
combined with cryopreservation of the cells at high density by controlled-rate freezing enables extensive quality control of the
cells before they are used for screening (upper right and middle right). It will also minimize variation in the screens that oth-
erwise would be present due to variations in cell quality/status. PBS, phosphate-buffered saline.

What are the critical factors for healthy cell suspensions
while plating during screens—proper pH, temperature,
time in suspension, oxygenation, etc.?
You can plate them in a water-jacketed spinner bottle
and hook that up to recirculating troughs on the automa-
tion platform. If it were a matter of seeding out cells to
preserve homogeneity between the first plate and the last
plate, then we would use the Ϫ80°C carrier on our plat-
form and cryogenically preserve the suspension cells at
high density. We can set up smaller batches of plates on
the platform and when a batch is done seed out more
cells. One vial of frozen cells can cover about 30 plates,
and we can have up to 12 vials on a platform. The incu-
bator can only accommodate 220 plates at a time, so we
have overcapacity when it comes to cryogenically pre-
served cells on the platform.
Proper pH, temperature, and oxygenation are all dic-
tated by the incubators. Cell seeding is a fast process, so
we do not worry too much about the physical conditions
while we seed out the cells. If we work with suspension
cells, the time in suspension is irrelevant. But if we use
adherent cells that are trypsinized and kept in a spinner
bottle as a source of cells for seeding, then we are lim-
ited to about 2 h before we start to induce apoptosis. Non-
suspension cells cannot be kept indefinitely in a spinner
bottle.
What are the critical factors in maintaining assay sensi-
tivity over cell passages (e.g., avoiding confluency, min-
imizing the number and extent of trypinizations, allow-
ing sufficient recovery of cells following plating before
stimulation, etc.)?
We do all of those types of studies as part of the as-
say development process. We know, for example, that if
we go above passage 8 or 9 with the HUVECs, they start
to lose response to many of the ligands we use for stim-
ulation. We prefer to use passage 6 or 7. We are now in
the process of implementing a diabetes-related assay that
includes three different stably integrated factors. We
know that if we let them go for about 10 passages, the
gene expression response goes down quite dramatically.
We have to use passage 3 or 4 to get a good response.
Part of assay development is determining what passage
is optimal to get the response you need.
What efforts do you take to detect and prevent
Mycoplasma contamination?
First, we purchase certified HUVECs from Cambrex.
We expand them very rapidly and freeze them down. If
we would see that the effect of an assay goes down, we
would have a direct indication that something is wrong
with the cells. We also have checkpoints to test for con-
tamination, and we recently implemented an in-house
polymerase chain reaction-based Mycoplasma test. If we
detect contamination, then of course we have to discard
the whole batch of cells.
For those who wish to adopt the tissue culture paradigm
you have developed, can you comment in general on how
cells grown on microcarrier beads subsequently behave
when plated onto plastic microtiter plates? Is this pro-
cess best used with a limited set of cell lines or can a va-
riety of cells be processed on microcarrier beads and
transferred to plastic surfaces?
Cells cultured on microcarriers have the same charac-
teristics when it comes to subsequent culturing on stan-
dard cell culture plastic as they would have if you were
to expand them in regular cell culture vessels (plastic).
A large variety of cells can be cultured in this fashion.
Human embryonic kidney 293 cells and other cells that
adhere poorly might be the problematic ones.
Parallel processing of multiple cell lines is restricted
to the number of spinner bottles you can connect to your
system, and that is dependent on the number of con-
trollers that you have access to. The controllers can eas-
ily be daisy-chained together. At present we would be
able to parallel process four separate cell lines if we had
three more controllers. The software is already set up for
that.
Has your group achieved the goal set of being able to
test 100,000 or more different genes in a single day? (If
not, what challenges remain?)
We are trying to establish the genomic reagents at
present. The limiting factor is the automation. Testing
100,000 genes in a single day would mean that if you
could transfect the genes into cells, you would then be
able to run them on multiple assays. For example, if you
were looking at 20 different genes, then you would have
to run them on five different assays to achieve testing
of 100,000 genes. It would not be a problem for us to
do that in one day. The challenge that remains is to es-
tablish all the necessary genomic reagents in the proper
format.
How did your dissertation work on developing synthetic
gene delivery systems, under Professor Edvard Smith at
the Karolinska Institutet in Sweden, lead to your position
at Columbia and the research you are now pursuing?
I knew that I wanted to study gene therapy when I was
12 years old. I read about retroviruses, which are nature’s
way of inserting a gene of interest into a cell. I wondered
whether retroviruses could be used to insert new genes
to correct diseases. This is still a viable concept. People
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tried to take this concept into the clinic too early on, with-
out knowing exactly what was going on at the genetic
level, and unfortunately this led to at least one patient
death.
Retrovirus elements are recognized as foreign by the
human immune system. So, if you use a retrovirus ele-
ment to drive gene expression you will get methylation
of those elements, which will shut down gene expression.
If you use non-retroviral vectors, such as adenovirus vec-
tors, you will have a problem with immune response. Be-
cause adenoviral vectors would yield transient expres-
sion, you would have to re-transduce the patient, which
would trigger an immune response.
I read a lot about gene delivery techniques and gene
therapy when I was younger—I was not your typical
teenager. I traveled in the United States and visited New
York for the first time with my cousin when I was 15
years old. After finishing my high school degree in Swe-
den I spent almost two years in the Swedish military and
then started my university studies in southern Sweden. I
focused on biology and genetics, with a lot of chemistry
also. During my last half-year of studies I approached a
Principal Investigator at the Karolinska Institute, Edvard
Smith, who had started experimenting with gene therapy.
He suggested that I also study immunology, and that is
what I focused on my last half-year. I then pursued my
Ph.D. with Edvard Smith, and I essentially wrote my own
Ph.D. program in gene therapy. He was very supportive.
He sent me to work with Donald Kohn at Children’s Hos-
pital in Los Angeles, who taught me how to make the
retrovirus vectors.
I worked on the concept of treating X-linked agam-
maglobulinemia (XLA) using gene therapy. It turned out
that to correct XLA, which is caused by a deficiency in
the Bruton’s tyrosine kinase, you need to introduce a va-
riety of control elements, and you cannot fit all the nec-
essary regulatory elements into a retrovirus. I was doing
this work in mice and nothing was working. After two
years, I began to look for a new gene delivery system
and invented Bioplex, or biological complexes, for gene
delivery.
Did this lead you to co-found Avaris, the company you
started while completing your Ph.D. work?
In the process of doing all these transductions with retro-
viruses I noticed that sometimes I would get really bad
titers, and you want as efficient gene delivery as possible.
I invented a device, basically a bioreactor that enhanced the
transduction efficiency by about 10-fold. That was about
the same time I invented these biological complexes. You
can read about this in an article by Brandén and Smith in
Methods in Immunology.1 Those patents became the basis
of the company. In Sweden you have the advantage of hav-
ing the company activities go on side-by-side with the work
of your academic laboratory In that way you can capital-
ize on the space and the equipment. You just pay a nomi-
nal fee for the use of the equipment and the space.
After some time I wanted to expand my experience,
and I had always wanted to live in New York City. I
wanted to work outside the field of gene delivery and
gene therapy as I was starting to get bored. Boredom is
my main enemy. I sent out two letters, one to James Roth-
man, who responded rather quickly and invited me to Co-
lumbia to give a seminar. I went in June 2002, gave a
seminar, had an interview with Jim, and met the post-
docs in the lab. I moved to New York City later that sum-
mer to begin work on a proteomics project Jim was run-
ning at Sloan Kettering. My job was to find problems in
the project and fix them. After about a year and half we
started talking about moving over to Columbia, and Jim
offered me the position as Associate Director of the Ge-
nome Center. He subsequently offered me the opportu-
nity to become Director of the MLSCN project.
—Interview by Vicki Glaser
Reference
1. Brandén LJ, Smith CIE: Bioplex technology. A novel, syn-
thetic gene delivery system based on peptides anchored to
nucleic acids. Methods Enzymol 2002;346:106–124.
Profile 471

Yale Center for High Throughput Cell Biology
P.O. Box 27381
West Haven, CT 06516
www.yale.edu/htcb
Center Infrastructure
The Yale Center for High Throughput Cell Biology utilizes Tecan Freedom EVO 200 platforms --
highly advanced, fully automated liquid and plate handling platforms capable of processing 96- to
1536-well plates. Our platforms can deliver liquid volumes from 10 nanoliters to 125 microliters and
can process over 200,000 wells per day. Barcoded screening plates allow digital plate tracking and
real-time data streaming to our enterprise-scale sample, plate, and results database. Three platforms
are housed in custom biosafety enclosures allowing for processing of BSL-2 samples including human
cells and certain pathogens. A fourth platform is used exclusively for immunostaining and aids in
maintaining high throughput.
The Center utilizes a Perkin Elmer Opera confocal imaging system containing four laser excitation lines
(405, 488, 561, and 635 nm) for high-content image acquisition. Capable of acquiring over 100,000
image sets per day, the Opera provides the Center with high throughput image acquisition capabilities
with integral image analysis scripting. With its Microlens-Enhanced Nipkow Disk, the Opera is
capable of imaging optical sections as thin as 1.3 µm for 3-dimemsional imaging. 4-D imaging is
accomplished using the Perkin Elmer UltraVIEW system. Designed for low throughput high-content
data acquisition the UltraVIEW system uses up to six laser lines to provide detailed images of expression
pattern changes through time. Fluorescence-based assays are read using Infinite Plate Readers integral
to the automation platforms.
The Center has deployed a highly scalable bioinformatics hardware infrastructure housed within a
modern industrial-scale data center. The data center is supported by layered security (physical,
hardware, software), redundant power supplies, and advanced environmental controls. The Center has
deployed databases and data processing applications that, for the purposes of high content imaging and
analysis, primarily utilize a range of high-performance IBM servers. Auxiliary computational resources
are available for high-throughput image analysis via a Center-administered Linux cluster as well as the
Yale High Performance Computing Center, the latter a massive cluster of over one thousand CPUs.
Computer disk space, always a concern with high content imaging, is available in ample quantities to
satisfy present and future needs; the currently deployed architecture is quickly scalable to 80 TB on
existing disk controllers and equipment, and can easily grow with minor enhancements.

P.O. Box 27381
www.yale.edu/htcb
Biological Profiling Assays
Yale Center for High Throughput Cell Biology (HTCB) has established core services group providing
low- and high- content screening for biopharmaceutical and academic partners. Utilizing expertise in
automation technology, high content imaging, and bioinformatics, the Center specializes in developing
and executing novel cell-based gene-splicing assays.
Assays Table
Type Assay Target Stimulation
Structural
p230 Golgi structure None
FAK FAK localization None
Phalloidin Actin structure None
Paxillin Paxillin localization None
Tubulin Tubulin structure None
Inflammation
E-selectin E-selectin expression
IL-1B, TNFa, CD40L, LPS,
Etoposide, None
IkB
IkBa (pSer32)
phosphorylation
TNFa, IL-1B, None
NFkB p65 translocation
Etoposide, None
NFkB (2)
NFkB (pSer 529)
phosphorylation
Etoposide, None
P-selectin P-selectin expression IL-4, None
VCAM VCAM expression
None
STAT Signaling
IRF9 IRF9 expression IFNg, None
STAT1
STAT1 pTyr 701
phosphorylation
IFNa, IFNg, None
STAT1 (2)
STAT1 pSer 727
phosphorylation
TNFa, IL-1B, None
STAT1 (3) STAT1 translocation IFNa, IFNg, None
STAT2 STAT2 translocation IFNa, None
STAT6 STAT6 translocation IL-4, None
Oxidative Stress HO1 HO1 expression None
PKC Signaling
PKCdelta
PKCdelta (pThr505)
phosphorylation
TPA, Etoposide
PKCmu
PKCmu (pSer916)
phosphorylation
TPA, None

P.O. Box 27381
www.yale.edu/htcb
Assays Table (continued)
Type Assay Target Stimulation
DNA Damage
Apoptosis
Cell Cycle
p53 Pathway
Alamar Blue Cytotoxicity None
ATM
ATM (pSer1981)
phosphorylation
Hydroxyurea, Etoposide,
None
Caspase3
Caspase 3 (Asp175)
cleavage
Staurosporine, None
CHK1
CHK1 (pSer345)
phosphorylation
None
CHK2
CHK2 (pThr68)
phosphorylation
None
Histone H2A.X
Histone H2A.X (pSer139)
phosphorylation
None
Histone H3
Histone H3 (pSer10)
phosphorylation
None
HSP27
HSP27 (pSer82)
phosphorylation
Anisomycin, Etoposide,
None
p53
p53 (pSer15)
phosphorylation
None
PKC Signaling
PKCdelta
PKCdelta (pThr505)
phosphorylation
TPA, Etoposide
PKCmu
PKCmu (pSer916)
phosphorylation
TPA, None
MAP Kinases
ERK1/2
ERK1/2 (pThr202/Tyr204)
phosphorylation
TPA, None
JNK
JNK (pThr183/Tyr185)
phosphorylation
Anisomycin, TNFa, IL-1B,
None
JNK (2) JNK localization
None
MAPKAPK2
MAPKAPK2 (pThr334)
phosphorylation
Anisomycin, None
MAPKAPK2 (2)
MAPKAPK2 (pThr222)
phosphorylation
Anisomycin, None
MKP1
MKP1 (pSer359)
phopsphorylation
TNFa, IL-1B, TPA, None
MNK1
MNK1 (pThr197/202)
phosphorylation
TNFa, IL-1B, TPA, None
MSK1
MSK1 (pThr581)
phosphorylation
TPA, None
p38
p38 (pThr180/Tyr182)
phosphorylation
Etoposide, None
Protein Synthesis
p70-S6K
p70 (pThr389)
phosphorylation
Anisomycin, Hydroxyurea,
None
p90-RSK
p90 (pThr360/Ser364)
phosphorylation
TPA, None
S6 ribosomal protein
S6 (pSer235/236)
phosphorylation
Anisomycin
If you are interested in an assay not listed, please feel free to contact us to discuss assay development
appropriate to your research.

P.O. Box 27381
www.yale.edu/htcb
Biological Profiling & Counter Screening
Yale Center for High Throughput Cell Biology (HTCB) has developed an extensive set of secondary
assays to counter-screen hits from primary screens to provide a deeper understanding of targets and
pathways, mechanisms of drug efficacy and toxicity, and ultimately human variation in therapeutic
response. In general, for phenotypic assay development, we have used a single cell type, HUVEC (human
umbilical vein endothelial cells). These cells have the advantages of being non-transformed and of human
origin, thus ensuring the biological validity of the assays. In addition, endothelial cells, which in vivo are
situated at the junction between the circulatory system and the solid tissues of the body, play an
active role in assimilating and transmitting signals. In this respect, it is noteworthy that endothelial
cells have been shown to possess a broad assortment of receptors capable of triggering many of the key
intracellular signaling pathways.
As shown in the table below, our repertoire of secondary assays monitors a variety of physiological states
and signaling pathways. Technically, these assays are divided between those that employ antibody
detection of a target protein and those that monitor transcriptional activation using luciferase reporter
gene readout. Many of the assays monitor specificity of kinase or transcription factor activation. Other
assays flag compounds that are toxic in some way or that cause a stress response in the cells.
Selected Counter-Screening Assays developed by Yale University Center for
High Throughput Cell Biology
Growth Factors Cytokines Apoptosis Cell Cycle Stress
Structural
Integrity
AP1 RE cJun NFAT RE Caspase 3 Cell Division
ATF6
RE
Actin
ATF2 MapkapK2 JNK Caspase 6 CHK2 ARE Fak
CRE Mnk1 STAT1 Caspase 7 cMyc DRE
P230
(Golgi)
CREB p38 STAT2 Caspase 8 Histone H3 Y-BOX
E2F RE PKCµ STAT6 HistoneH2a ThRE
ERK1/2 PMA RE NFkB ATM HO1
cFos cSrc E-Selectin p53
Hsp27 SRE VCAM-1
JNK(1) C-MARE
Abbreviations used: RE, response element; CRE, cAMP response element; CREB, cAMP response element binding
protein; SRE, serum response element. ARE, antioxidant response element; NFAT, nuclear factor of activated T
cells; DRE, dioxin response element; C-MARE, CRE-like maf response element; NFkB, nuclear factor kB; HO1,
heme oxygenase-1; ThRE, Thrombin response element.

P.O. Box 27381
www.yale.edu/htcb
We are also incorporating over 50 additional assays based on adenoviral vectors into our array of profiling
assays. Of these, 25 assays use a fluorescent protein reporter gene to monitor protein translocations and
require an image-based readout suitable for multiplexing. Another 20 assays feature and enzymatic
reporter to measure transcriptual activation from a variety of response elements.
SID 855810 SID 855758 SID 856923 SID 857745 SID 858409 SID 861918
An example of compound activity in 42 secondary assays
Red indicates inhibition; blue activation. Each compound was tested at 10 serial 2-fold dilutions with the
highest concentration (10 µM) on the left. The overall inhibition level of all the assays, in red above,
indicates the level of promiscuity of each of the assay compounds.
O
OH
H
H
O
NNH
OHO
O
N
N
O
OH
O
OO
O
O
ONH
SO
O
N N OSHO
N
NH
N
N
N
O
O
N
N
N
NS
NFkB TNFa
NFkB TNFa
NFkB IL-1B
NFkB IL-1B
E-selectin IL-1BE-
VCAM TNFa
VCAM IL-1B
p38 Anis
p38 IL-
ERK1/2 TPA
STAT1 IFNg
STAT6 IL-4
JNK Anis
JNK TNFa
JNK IL-1B
p53 No ne
p230(Agrains) No ne
Histone H3 No ne
Tubulin (4hr) No ne
Cell Num ber (4hr) No ne
Cell Number (24hr) No ne
AP1 PMA
PMA PMA
NFAT PMA
E2F PMA
Rb PMA
CRE PMA
ATF6 No ne
ARE No ne
ThRE No ne
Y-BOX No ne

P.O. Box 27381
www.yale.edu/htcb
High Content Screening
The Yale Center for High Throughput Cell Biology has established a core services group specializing in
high content screening and other technologies. Utilizing expertise in automation technology, high content
imaging, and bioinformatics, the Center develops and executes novel cell-based assays. The Center
provides flexible cost-effective solutions to biopharmaceutical and academic partners and provides novel
insights about biological phenomena.
Whenever feasible, we multiplex readouts for each assay used as a primary screen. This increases the
depth of the data collected as well as the biological significance of the data. Our imaging platform, the
Perkin Elmer Opera, can utilize up to three different laser lines, in addition to UV excitation, for either
sequential imaging or simultaneous imaging of multiple channels.
For most assays, one channel is dedicated to visualizing the cells’ nuclei. Thus all image-based assays are
already multiplexed for cell number and nuclear size and shape – useful controls as decreased numbers or
condensed nuclei can be indications of toxicity or apoptosis. The majority of image-based cellular assays
use either a fluorescent protein, such as GFP, or antibodies coupled to fluorescent markers to visualize
cellular components. In designing a multiplexed assay, the technical requirements include having
fluorescent readouts that are on different channels (e.g. red and green) and that have well separated
spectra of emitted light.
As an example, the first assay that we multiplexed was VCAM-1, which was screened against a 100k
compound library. During inflammation, cytokine activation of the NFkB pathway in vascular endothelial
cells results in cell surface expression of VCAM-1. Adhesion and subsequent transmigration of circulating
monocytes is carried out by VCAM-1 anchored to the cytoskeleton by its cytoplasmic domain. Following
migration out of the lumen, monocytes are transformed into macrophages that can accumulate lipid, a
critical early step in the development of atherosclerosis1, 2
. Thus, it is possible to block monocyte adhesion
by two independent routes, decreased VCAM-1 expression or disruption of F-actin fibers3, 4
. A recognized
anti-inflammatory agent, simvastatin, inhibits monocyte adhesion despite increased VCAM expression
because it simultaneously disorganizes the cytoskeleton5
1
O’Brien, et al., 1996, Circulation (93), 672
2
Ross, 1999 N Engl J Med (349), 115
3
Wojciak-Stothard, et al., 1999 J Cell Physiol (176), 150
4
Vandenberg, et al., 2004 J Cell Biochem (91), 926
5
Pozo, M., et al., 2006, Eur J Pharm (548), 53
. Clearly, a screen for small molecules that block
monocyte adhesion to Human Umbilical Vein Endothelial Cells (HUVEC) would have both biological
and therapeutic significance. However, such a screen would be difficult and costly to perform in a high

P.O. Box 27381
www.yale.edu/htcb
throughput mode. Fortunately, similar results can be obtained by multiplexing the assay for VCAM-1
expression with detection of F-actin. The actin cyctoskeleton is easily visualized using phalloidin, which is
specific for polymerized actin, (i.e. F-actin). Phalloidin, which is read in the green channel, is comparable
with visualization of VCAM-1 using an antibody visualized in the red channel. For a multiplexed VCAM-
1 assay, Human Umbilical Vein Endothelial Cells (HUVEC) are dosed with compounds, stimulated with
IL-1b, and 24 h later fixed and stained for VCAM-1 and F-actin fibers.
Multiplexed VCAM-1 assay monitors
VCAM-1 expression and actin cyctoskeleton
These images provide information such as:
• Nuclear shape (the size, blebbing) which can be an indication of apoptosis and
nuclear fragmentation;
• The general morphology of cells which can indicate stress and show blebbing,
which may indicate apoptosis
• Shape and appearance of the cytoskeleton (visualization by phalloidin staining)
can indicate whether there is a problem with the actin polymerization or a more
general problem with the cytoskeleton; and
HUVEC, 24 h after treatment with TNFa 10ng/ml, and stained with anti-VCAM-1 (A and B)
and phalloidin (A and C)

P.O. Box 27381
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• The level and location of the VCAM-1 which can indicate protein distribution
problems in the intracellular transport machinery (such as vesicular transport).

P.O. Box 27381
www.yale.edu/htcb
Informatics
Informatics at The Yale Center for High Throughput Cell Biology (HTCB) is redefining modern
genomics analysis. As a vital element of modern life sciences research, informatics is fundamental to our
Center’s mission of providing world-class, publication-level results.
Genome-wide RNAi analysis at HTCB is enriched by our inherently collaborative processes. Our
informaticians and biologists focus on understanding the research objectives of our collaborators. In
order to translate experimental results into broad biological context, our informatics scientists study
relevant cellular pathways of interest in silico, apply novel methods to discover relevant marginal hits
from our in vitro screens, and fit confirmed gene hits into broader protein interaction networks. As a
result, we can help prioritize high impact follow-up experiments.
HTCB Informatics also provides expert level cheminformatics support, exploiting the synergies between
compound profiling results (chemical activators and inhibitors) and gene hits from RNAi campaigns.
Combining data sets, we can overlay novel screening data with, for example, known protein/tissue
interaction data and can speed the discovery of novel biomarkers.
We have developed unique approaches to exploring hit-space via our novel software development
efforts; via machine learning and other techniques we uncover the biological significance of subtle
patterns of gene activity across seemingly disparate systems.
Advanced analyses are available that focus on subjects of highest interest to our collaborators. Advanced
techniques can include access to custom databases and tools specifically scripted per project.
Hit-space analysis:
• Clustering and annotation
• Inferred hit analysis
• Pathways analysis
• Protein network analysis
• Disease interactions
• Chemical interactions
• Tissue interactions
Profiling analysis:
• Chemical epistasis analysis
• Gene profiling analysis
High content analysis:
• Image analysis script authoring and
optimization
• Multiparametric analysis

P.O. Box 27381
www.yale.edu/htcb
Selected Assay Publications
IL-1B Induced NFkB Translocation
Keywords: high content, cell-based assay, primary screens
http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=796
Chemical Inhibitors of TNF Alpha Stimulated VCAM1 Expression
Keywords: high content, multiplexed, cell-based assay, biological signal transduction
Chemical Modifiers of Cytoskeleton Assembly
Keywords: high content, multiplexing, microscopy
Clearance of Mutant Huntintin Protein
Keywords: high content, cell-based assay, neurodegenerative disease mode/poly Q
Novel sHE Inhibitors for the Therapeutic Treatment of Hypertension
and Inflammation
Keywords: enzymatic assay, biochemical assay, low-content
LYP Inhibitors-An Autoimmunity Target
Keywords: enzymatic assay, biochemical assay, low-content

P.O. Box 27381
www.yale.edu/htcb
siRNA Genome-wide Screening
The Yale Center for High Throughput Cell Biology has established a core services group specializing in
genome-wide RNAi screening. Utilizing expertise in automation technology, high content imaging and
bioinformatics, the Center develops and executes novel cell-based gene-silencing assays. The flexible,
automated infrastructure allows for high throughput RNAi screening of various assay formats. The
Center provides cost-effective solutions to biopharmaceutical and academic partners and provides novel
insights about biological phenomena.
We collaborate closely with principal investigators from the initial assay design phase through
confirmation and characterization of hits. We create assays capable of interrogating biological pathways of
interest and screen them against the Dharmacon siGenome library. Using our automation platforms we
conduct genome wide screens at high efficiency and with high precision. Project objectives can be met by
single parameter plate reader assays for endpoint detection such as luminescence, fluorescence,
absorbance, and FRET. Using the Opera confocal imager, we capture and analyze multi-parametric data
sets to measure biological events such as:
• Phosphorylation
• Translocation
• Change in expression
• Redistribution
• Co-localization
Our imaging capabilities allow us to multiplex up to three different primary readouts as well as secondary
readouts such as cell number and morphology. We have four laser lines at present, 405, 488, 561, 640
measuring from UV to far red. Customized image algorithms offer multiple analysis options that allow
for alternative hit classification and definitions.
We offer a suite of profiling assays designed to further elucidate the mechanisms of action of the hits
generated in the primary screen. Our assays cover a range of areas and can be used to rule out non-specific
hits to help focus follow-up work. We can additionally design assays tailored to areas of interest in the
given pathway. Using existing knowledge in the field we can choose key areas of regulation for assay
development and provide an insight into which part of the pathway is being affected and at what level.
Our bioinformatics tools have been developed to utilize screening campaign results to characterize genes.
Our goal is to systematically discover functionality shared by genes by identifying similar activity profiles
between genes screened across our wide range of cellular assays.
We encourage interested parties to contact us to discuss opportunities or to schedule a tour of our
facilities. Please contact Dr. Lars Branden at the Yale Center for High Throughput Cell Biology at
lars.j.branden@yale.edu for further information.

P.O. Box 27381
www.yale.edu/htcb
Pricing and Expertise Guide
The Yale Center for High Throughput Cell Biology is pleased to offer low and high-content high
throughput screening to corporate and academic partners. Pricing for screening campaigns is highly
variable, depending on conditions such as preexisting high-throughput assay readiness, number and
type of cellular markers used, and informatics needs. Our pricing structure is based on a flat fee for
library access and literature reviews with variable costs for laboratory consumables, assay development
and informatics labor. We encourage interested parties to contact the Center’s Director, Dr. Lars
Branden, lars.j.branden@yale.edu for more information on services and fees.
Every service that Yale HTCB provides has an intrinsic and vital component of bioinformatics
incorporated. HTCB informatics experts provide support including sample tracking, data capture, data
storage, data analysis, backup, and retrieval. The close collaboration between Center biologists and
informaticians allows for iterative analysis of screening data sets, thus providing Center collaborators
deeper insights into the systemic biological meaning of screen results.
Our expertise includes:
• Researching, preparing, and executing high-content screens
• Developing assay descriptions/protocols
• Optimizing assays for high-throughput screening
• Process flow
• Consulting on assay development
• Biological profiling & analysis of genes
• Defining biological activity of chemical analogs
• Preparing literature reviews on relevant areas
• Library reformatting
• Bulk production of cells/viruses
• Aliquoting
• Screening consultancy

Assay and Drug Development Technologies, Vol.5 Number 4, 2007

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