Western Blotting Tips

1) Load 20 to 35 μl of your samples unless you know for a fact that this is too much.

2) In the first lane load 25 μl of positive bloting control. This is lysate stimulated with PMA or LPA. This is good for most CCL39 blots.

3) Molecular Wt Markers - There are now two kinds. One is the BioRad Prestained standards we've always used. The other is MagicMarker. This is a non-stained protein that has an IgG recognition site. That means each of the proteins will bind secondary antibody and will be visible on the film. To use this:

- In the second lane load 10-15 μl of the BioRad standard

- In the SAME well/lane add 3 μl of the MagicMarker standard (found in a box in the

freezer in the main room

4) Run the gel to the bottom but not off. Make certain the dye is just coming to the edge of the glass not the gasket. Remember, 200 v for 1 hour is an approximation - you may very likely have to turn it up a little more. If this is a key experiment, run the gel at 100 v for 10-15 min first then run for 200V. The proteins will be tighter. ALSO if the % cross-linking is low then the proteins will run faster than the higher % gels

5) Soak the gel in transfer buffer for a minute or two to reduce salt that can reduce transfer.

6) Cut the paper to fit the gel, unless you are using ERK or p-ERK. Remember unless we have done the blot many times you need to show the molecular weights to prove that the band you think is your protein is the right one. Clip the corner of the blot as directed in the protocol. Not all antibodies are as specific as some of the ones we use. Always mark the film with the markers!

7) The fit of the gel sandwich should be tight and free of air bubbles. If the pads are getting

thin add a third to make certain there is proper contact between paper and gel.

8) Transfer at 90 V for 1 hr unless using a 12-16% gel then add 15 min

9) Some antibodies need a different washing solution. The concentration or type of detergent (Tween-20) may not work for some. If using a new antibody, check!

10) Block in the BioRad dry milk, not the carnation or other generic instant milk. Use the same concentration as before. We get high backgrounds with some of these other milks. Some antibodies need BSA instead of milk to block with. Always look up new antibody requirements.

11) When starting use 1:500 dilutions for the primary and 1:1000 for the secondary. Use only the antibodies from the "Chinese take-out" cool-safe box

12) Don't let the blot get too dry - this may have been a problem

13) Do a 10 sec, 1 min and 10 min exposure.

Purifying DNA from TAE agarose gels

1. Excise band from agarose gel.

2. Add 3 volumes of NaI and incubate at 55°C to melt gel.

3. Add 5μl GLASSMILK suspension.

4. Pellet GLASSMILK/DNA complex (5 seconds).

5. Wash pellet with 1ml NEW Wash.

6. Pellet again and rewash with 0.5ml NEW Wash.

7. Elute DNA with 10-20μl water or 0.1x TE.

Proteolytic enzymes

Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum.

Lipid siRNA transfection

For some cell lines, either electroporation or cationic lipids may be used. Basically any tfx protocol will require you to work out conditions for every cell line. Sometimes between 70-90% or more cells can be transfected with cationic lipids. With a bit of tweaking, and depending on the cell line, you can drive siRNAs into almost 100% of the cells with the following protocol:

Seed cells high, so that they are 85%+ on the day of transfection:

For one 10 cm dish:

40 ul Lipofectamine 2000

20 ug DNA

100-300 pmoles siRNA

tube 1

add the siRNA and/or DNA to 2 ml SF-DMEM (no drugs added)

tube 2

add the lipid to 2 ml SF-DMEM (no drugs added)

mix these two tubes immediately, for a volume of 4 ml, and then add to cells which have been washed 2X with SF-DMEM. Incubate 3-6 hours, then add normal media. Check for expression or knockdown 36+ hours post-transfection. Knockdown by siRNAs usually last 3-5 cell doublings.

When oligofectamine is used, the identical protocol is used, but:

Seed cells at 15% confluency, perform tfx with 30 ul oligofectamine and siRNAs, then repeat tfx 36 hours later. Analysis is performed when cells are 85-90% confluent. I have not calculated the transfection efficiency with oligofectamine.

ELISPOT Protocol

Coat the Plate:
1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s
LEAF™ format antibodies are specifically designed for this assay) to a
final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100
μl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore;
Cat. No. MAIPS-4510).
2. Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in
humidified atmosphere.

Block the Plate:
3. Wash plate 3 times with sterile PBS, 200 μl/well.
4. Add 200 μl/well of sterile Blocking Buffer.
5. Seal plate and incubate at room temperature for ≥ 1 hour.
6. Wash plate 3 times with sterile PBS, 200 μl/well.
Set-Up Tissue Culture and Add Antigen or Mitogen:
7. Add appropriate sterile antigen or mitogen solution diluted in appropriate
sterile tissue culture medium (TC) to ELISPOT plate, 100 μl/well.
8. Add cells diluted in sterile TC medium, 100 μl/well. Use 50,000-500,000 cells/
well (the minimum number of cells should be determined in preliminary
experiments).
9. Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the
optimum stimulation period. BioLegend recommends a 24 hour incubation
for IFN-γ, IL-2, and TNF-α, and a 48 hour incubation period for IL-4, IL-5, and
IL-10 for most activation conditions.

Add Detection Antibody:
10. Wash plate 3 times with PBS, 200 μl/well.
11. Wash plate 3 times with PBS-Tween, 200 μl/well.
12. Add 100 μl/well of diluted biotinylated detection antibody at 0.25-2 μg/ml in
PBS-Tween-BSA.
13. Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature.

Add Avidin-Horseradish Peroxidase (Av-HRP):
14. Wash plate 4 times with PBS-Tween, 200 μl/well.
15. Add 100 μl per well of the Av-HRP conjugate (Cat. No. 405103) or other
enzyme conjugate diluted to its pre-determined optimal concentration in
PBS-Tween-BSA (usually between 1/500 – 1/2000).

Source: 1. postech.ac.kr

2.http://www.biolegend.com/media_assets/support_protocol/BioLegend_ELISPOT_protocol.pdf

Competitive ELISA Protocols

AbVideo™ - Competitive ELISA

3 May 2010 ... Competitive ELISA is through competitive binding to measure the amount of analyte. First of all, unlabeled antibody is incubated in the ...
www.abnova.com › Support › AbVideo™

Millipore - Competitive ELISA assays, Competitive ELISA protocol.

Competitive ELISA assays, Competitive ELISA protocol and Competitive ELISA method - General Protocol for the Competitive ELISA Method.
www.millipore.com/cellbiology/.../competitiveelisamethod

Clinical utility of a competitive ELISA to detect antibodies ...
Clinical Utility of a Competitive ELISA. 85 lines for their use as a screening test. Following the comple- tion of the present evaluation, we proposed a ...
www.ugr.es/~cts521/documentos/Gutier-JClinLabAnal14.pdf

Competitive ELISA
In the competitive ELISA, two antigens are involved; one, the sample antigen to be assayed and the other, a constant level of "binding" antigen immobilized ...
www.springerlink.com/index/urh3412h3612g473.pdf

Competition Elisa Protocols | eHow.com

7 Dec 2010 ... Competition Elisa Protocols. The ELISA test is an enzyme-linked immunosorbent assay that uses antibodies or antigens coupled to a specific ...
www.ehow.com › ... › Science & Nature › Science

Springer Protocols: Competitive ELISA

2 Direct Competitive ELISA ... 2.1 Learning Principles; 2.2 Reaction Scheme; 2.3 Materials and Reagents; 2.4 Practical; 2.5 Competition Assay Prope. ...
www.springerprotocols.com/Abstract/doi/10.../0-89603-279-5:177

Competitive ELISA

Competitive ELISA is through competitive binding. The steps for this ELISA are somewhat different than the others:

1. Unlabeled antibody is incubated in the presence of its antigen (Sample)

2. These bound antibody/antigen complexes are then added to an antigen-coated well.

3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.

5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).

Commonly the antigen is not first positioned in the well. (Wikipedia)

Competitive Elisa Protocol

1. For most applications, a polystyrene microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

2. Add 50 µL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using a checkerboard titration prior to testing samples. PVC will bind approximately 100 ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 µg/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again. Allow to incubate for 4 hours. at room temperature or 4°C overnight. Note: If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody according to the following procedure: (1) Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 µL of antibody solution to each well (20 µg/mL in PBS). (2) Incubate the plate overnight at 4°C to allow complete binding. (3) Add primary capture antibody (as above).

3. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.

4. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs to overnight in a humid atmosphere at room temperature.

5. Wash wells twice with PBS.

6. Add 50 µL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20).

7. Note: Sodium azide is an inhibitor of horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.

8. Add 50 µL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hours. at room temperature in a humid atmosphere.

9. Wash the plate four times with PBS

10. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.

11. Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.

(Source: Millipore)

Cloning Fact Sheet (2)

What animals have been cloned?

Scientists have been cloning animals for many years. In 1952, the first animal, a tadpole, was cloned. Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. Since Dolly, researchers have cloned a number of large and small animals including sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur. All these clones were created using nuclear transfer technology.

Hundreds of cloned animals exist today, but the number of different species is limited. Attempts at cloning certain species have been unsuccessful. Some species may be more resistant to somatic cell nuclear transfer than others. The process of stripping the nucleus from an egg cell and replacing it with the nucleus of a donor cell is a traumatic one, and improvements in cloning technologies may be needed before many species can be cloned successfully.

Can organs be cloned for use in transplants?

Scientists hope that one day therapeutic cloning can be used to generate tissues and organs for transplants. To do this, DNA would be extracted from the person in need of a transplant and inserted into an enucleated egg. After the egg containing the patient's DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested. The stem cells would be used to generate an organ or tissue that is a genetic match to the recipient. In theory, the cloned organ could then be transplanted into the patient without the risk of tissue rejection. If organs could be generated from cloned human embryos, the need for organ donation could be significantly reduced.

Many challenges must be overcome before "cloned organ" transplants become reality. More effective technologies for creating human embryos, harvesting stem cells, and producing organs from stem cells would have to be developed. In 2001, scientists with the biotechnology company Advanced Cell Technology (ACT) reported that they had cloned the first human embryos; however, the only embryo to survive the cloning process stopped developing after dividing into six cells. In February 2002, scientists with the same biotech company reported that they had successfully transplanted kidney-like organs into cows. The team of researchers created a cloned cow embryo by removing the DNA from an egg cell and then injecting the DNA from the skin cell of the donor cow's ear. Since little is known about manipulating embryonic stem cells from cows, the scientists let the cloned embryos develop into fetuses. The scientists then harvested fetal tissue from the clones and transplanted it into the donor cow. In the three months of observation following the transplant, no sign of immune rejection was observed in the transplant recipient.

Another potential application of cloning to organ transplants is the creation of genetically modified pigs from which organs suitable for human transplants could be harvested . The transplant of organs and tissues from animals to humans is called xenotransplantation.

Why pigs? Primates would be a closer match genetically to humans, but they are more difficult to clone and have a much lower rate of reproduction. Of the animal species that have been cloned successfully, pig tissues and organs are more similar to those of humans. To create a "knock-out" pig, scientists must inactivate the genes that cause the human immune system to reject an implanted pig organ. The genes are knocked out in individual cells, which are then used to create clones from which organs can be harvested. In 2002, a British biotechnology company reported that it was the first to produce "double knock-out" pigs that have been genetically engineered to lack both copies of a gene involved in transplant rejection. More research is needed to study the transplantation of organs from "knock-out" pigs to other animals.

What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90% of cloning attempts fail to produce viable offspring. More than 100 nuclear transfer procedures could be required to produce one viable clone. In addition to low success rates, cloned animals tend to have more compromised immune function and higher rates of infection, tumor growth, and other disorders. Japanese studies have shown that cloned mice live in poor health and die early. About a third of the cloned calves born alive have died young, and many of them were abnormally large. Many cloned animals have not lived long enough to generate good data about how clones age. Appearing healthy at a young age unfortunately is not a good indicator of long-term survival. Clones have been known to die mysteriously. For example, Australia's first cloned sheep appeared healthy and energetic on the day she died, and the results from her autopsy failed to determine a cause of death.

In 2002, researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, reported that the genomes of cloned mice are compromised. In analyzing more than 10,000 liver and placenta cells of cloned mice, they discovered that about 4% of genes function abnormally. The abnormalities do not arise from mutations in the genes but from changes in the normal activation or expression of certain genes.

Problems also may result from programming errors in the genetic material from a donor cell. When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryos.

Should humans be cloned?

Physicians from the American Medical Association and scientists with the American Association for the Advancement of Science have issued formal public statements advising against human reproductive cloning. The U.S. Congress has considered the passage of legislation that could ban human cloning.

Due to the inefficiency of animal cloning (only about 1 or 2 viable offspring for every 100 experiments) and the lack of understanding about reproductive cloning, many scientists and physicians strongly believe that it would be unethical to attempt to clone humans. Not only do most attempts to clone mammals fail, about 30% of clones born alive are affected with "large-offspring syndrome" and other debilitating conditions. Several cloned animals have died prematurely from infections and other complications. The same problems would be expected in human cloning. In addition, scientists do not know how cloning could impact mental development. While factors such as intellect and mood may not be as important for a cow or a mouse, they are crucial for the development of healthy humans. With so many unknowns concerning reproductive cloning, the attempt to clone humans at this time is considered potentially dangerous and ethically irresponsible.

(Source: www.ornl.gov)

Cloning Fact Sheet (1)

Introduction

The possibility of human cloning, raised when Scottish scientists at Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385, 810-13, 1997), aroused worldwide interest and concern because of its scientific and ethical implications. The feat, cited by Science magazine as the breakthrough of 1997, also generated uncertainty over the meaning of "cloning" --an umbrella term traditionally used by scientists to describe different processes for duplicating biological material.

What is cloning? Are there different types of cloning?

When the media report on cloning in the news, they are usually talking about only one type called reproductive cloning. There are different types of cloning however, and cloning technologies can be used for other purposes besides producing the genetic twin of another organism. A basic understanding of the different types of cloning is key to taking an informed stance on current public policy issues and making the best possible personal decisions. The following three types of cloning technologies will be discussed: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.

Recombinant DNA Technology or DNA Cloning

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning," and "gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.

Scientists studying a particular gene often use bacterial plasmids to generate multiple copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome (see image to the right). Plasmids and other types of cloning vectors were used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial plasmids, some other cloning vectors include viruses, bacteria artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Cosmids are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA and can be packaged in lambda phage particles for infection into E. coli cells. BACs utilize the naturally occurring F-factor plasmid found in E. coli to carry 100- to 300-kb DNA inserts. A YAC is a functional chromosome derived from yeast that can carry up to 1 MB of foreign DNA. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

Reproductive Cloning

Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.

Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

How can cloning technologies be used?

Recombinant DNA technology is important for learning about other related technologies, such as gene therapy, genetic engineering of organisms, and sequencing genomes. Gene therapy can be used to treat certain genetic conditions by introducing virus vectors that carry corrected copies of faulty genes into the cells of a host organism. Genes from different organisms that improve taste and nutritional value or provide resistance to particular types of disease can be used to genetically engineer food crops. With genome sequencing, fragments of chromosomal DNA must be inserted into different cloning vectors to generate fragments of an appropriate size for sequencing.

If the low success rates can be improved (Dolly was only one success out of 276 tries), reproductive cloning can be used to develop efficient ways to reliably reproduce animals with special qualities. For example, drug-producing animals or animals that have been genetically altered to serve as models for studying human disease could be mass produced.

Reproductive cloning also could be used to repopulate endangered animals or animals that are difficult to breed. In 2001, the first clone of an endangered wild animal was born, a wild ox called a gaur. The young gaur died from an infection about 48 hours after its birth. In 2001, scientists in Italy reported the successful cloning of a healthy baby mouflon, an endangered wild sheep. The cloned mouflon is living at a wildlife center in Sardinia. Other endangered species that are potential candidates for cloning include the African bongo antelope, the Sumatran tiger, and the giant panda. Cloning extinct animals presents a much greater challenge to scientists because the egg and the surrogate needed to create the cloned embryo would be of a species different from the clone.

Therapeutic cloning technology may some day be used in humans to produce whole organs from single cells or to produce healthy cells that can replace damaged cells in degenerative diseases such as Alzheimer's or Parkinson's. Much work still needs to be done before therapeutic cloning can become a realistic option for the treatment of disorders.

CAM ASSAY

Shell-less embryo culture

Fertilized white leghorn chicken eggs (SPAFAS Inc., Norwich, CT) were received at day 0 and

incubated for 3 days at 37°C with constant humidity. On day 3, eggs were rinsed with 70% ethanol and

opened into 100 mm2 tissue culture coated Petri dishes under aseptic conditions. The embryos were then

returned to a humidified 38°C incubator2 for 7-9 additional days.

Mesh assay

Substrates

Vitrogen (Collagen Biomaterials, Palo Alto, CA) was diluted to a concentration of 1.46 mg/ml with

an equal volume of DMEM (HEPES buffer, no phenol red, GIBCO BRL, Gaithersberg, MD). This 1:1 mixture

made up half of the total volume to be pipetted onto the mesh (final concentration = 0.73 mg/ml). Matrigel

(Becton Dickinson, Bedford, MA) was diluted to a final concentration of 10 mg/ml with DMEM and directly

pipetted onto meshes. Nylon mesh with 250 μm2 openings were cut into 4 mm x 4 mm squares and

autoclaved. In preparation for polymerization, meshes were placed, under aseptic conditions, onto a nonbinding

surface (i.e., bacteriological Petri dish). The polymerization conditions for each substrate were

identical; after mixing the growth factors and/or compounds, 40 μl were pipetted onto each mesh in a

bacteriological Petri dish as described above. The Petri dish was placed in a humidified 37°C incubator with

5% CO2 for 30 minutes to allow polymerization followed by an incubation at 4°C for 2 hours.

Growth Factors

VPF/VEGF165 (Peprotech, Rocky Hill, NJ) was resuspended at 100 ng/μl in HBSS (Sigma, St.

Louis, MO). For each mesh containing VPF/VEGF, 250 μg was used.

Placement of meshes

In a tissue culture enclosure, meshes were placed onto the periphery of the CAM of a day 12-14

embryo, excluding areas containing major vessels. The embryos were then returned to the humidified 38°C

incubator with 3% CO2 for 24 to 48 additional hours.

Visualization of vessels

Embryos were removed from the incubator and meshes were viewed under a dissecting

microscope for gross evaluation. For injection, borosilicate glass capillaries (OD 1.0 mm, ID 0.75 mm; Sutter

Instrument Company, Novato, CA) were prepared with P-87 micropipette puller (Sutter Instrument

Company). Needles were connected with Tygon tubing (ID 1/32", OD 3/32", wall 1/32") to a 3cc syringe

with a 20-gauge needle. The syringe was attached to an infusion pump (Harvard Apparatus, South Natick,

MA). Injection of 400 μl FITC dextran, MW 2,000,000 (Sigma, St. Louis, MO), into the umbilical vein was

performed at a rate of 200 μl per minute. The FITC dextran was allowed to circulate for 5 minutes and 3.7%

formaldehyde in PBS was applied directly on each mesh. The embryos were then incubated at 4°C for 5

minutes and the meshes were dissected off the CAM and fixed in 3.7% formaldehyde for 10 minutes to

overnight.

Quantification of vessels

After fixation, meshes were mounted on slides with 90% glycerol in 1 X PBS and visualized on an

inverted fluorescence microscope. A Nikon Diaphot with a Sony DXC-151A camera attached to the side

port was used for capture of images, which were transferred to a Power Macintosh 7100/66AV. NIH Image

1.61 (public domain program available on the Internet at http://rsb.info.nih.gov/nih-image/) was used to

capture and analyze images. For each mesh, 5 random staggered images (approximately 600 μm each) were

captured using "Capture Frames," followed by "Make Montage" in order to display all of the frames at once.

The areas of high intensity were highlighted using "Density Slice" with (LUT selected at 105) and measured

using the "Measure" function. When density slicing is used in an image, the "Measure" function calculates

the areas of highlighted pixels.

（Source: Arispe Lab）

Angiogenic Models-chorioallantoic membrane assay

Biomacromolecules - CAM preparation

CAM preparation. IPW research units. Prof. D. Neri · Prof. H. Wunderli - Allenspach · Prof. U. Spichiger - Keller · Prof. H.P. Merkle · Prof. U. Quitterer ...
www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/cam

Chorioallantoic Membrane Vascular Assay (CAMVA)

Chorioallantoic Membrane Vascular Assay (CAMVA). Theory: Vascular damage (i.e. ghost vessels, capillary injection, or. hemorrhaging) of the chorioallantoic ...
www.iivs.org/pages/methods/CAMVAsummarysheet.pdf

The gelatin sponge–chorioallantoic membrane assay

The gelatin sponge–chorioallantoic membrane assay. Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo ...
www.natureprotocols.com/2006/06/23/the_gelatin_spongechorioallant.php

THE CHICK CHORIOALLANTOIC MEMBRANE AS A MODEL TISSUE FOR SURGICAL Retinal Research Simulation.
factors in ocular tissues of normal rabbits on chorioallantoic. membrane assay. Tohoku J Exp Med 1988;154:63–70. 10. Prost M. Experimental studies on the ...
www.stanford.edu/~palanker/publications/CAM%20as%20retinal%20model.pdf

Chorioallantoic membrane assay: vascularized 3-dim.

PubMed is a service of the US National Library of Medicine that includes over 16 million citations from MEDLINE and other life science journals for ...
www.ncbi.nlm.nih.gov/pubmed/11547121

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay.

Assessment of Angiogenic Factors The Chick Chorioallantoic Membrane Assay. By: Adam Jones3, Chisato Fujiyama3, Stephen Hague3, Roy Bicknell4 ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-137-X:119

Chicken chorioallantoic membrane assay (Cytokines & Cells ...

Chicken chorioallantoic membrane assay. The following COPE entries contain this entry term or one of its hypertext synonyms: ...
www.copewithcytokines.de/cope.cgi?key=Chicken%20chorioallantoic%20membrane%20assay

THE SHELL-LESS CHIC CAM ASSAY: A MODEL FOR STUDYING MELANOMA

ANGIOTROPISM AND EXTRAVASCULAR MIGRATORY METASTASIS
http://www.med.miami.edu/mnbws/documents/06Lugassy.pdf

Chorioallantoic Membrane Vascular Assay - Alternative Ocular Irritation Testing

Chorioallantoic Membrane Vascular Assay Alternative Ocular Irritation. Day 7 - Vascular development of the Chorioallantoic Membrane. ASSAY HIGHLIGHTS: ...
www.camva.com/

The gelatin sponge-chorioallantoic membrane assay.

The gelatin sponge–chorioallantoic membrane assay ... quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) ...
www.nature.com/nprot/journal/v1/n1/abs/nprot.2006.13.html

angiogenic models

Classical angiogenesis assays include the chick chorioallantoic membrane, rabbit cornea assay, sponge implant models, matrigel plugs and conventional tumor ...
www.med.unibs.it/~airc/sandra/models.html

Evaluating Compounds Affecting Angiogenesis.indd

ASSAY. In a typical angiogenesis evaluation using the CAM assay,. fertilized chick eggs are incubated at 37°C and specific. humidity (60%) for 3 to 4 days. ...
www.sri.com/biosciences/pdf/EvaluatingCompoundsAffectingAngiogenesis.pdf

Animal models Protocols

A list of animal model protocols from scientific journals, patents and/or laboratory websites.

Animal Model for Assessing Therapeutic Efficacy of .alpha.-Amino Acid Prodrugs for Treating Alzheimer's Disease. US Patent#7,531,572 Protocol

Animal Model for Assessing Therapeutic Efficacy of .alpha.-Amino Acid Prodrugs for Treating Huntington's Disease. US Patent#7,531,572 Protocol

Animal Model for Assessing Therapeutic Efficacy of .alpha.-Amino Acid Prodrugs for Treating Spasticity. US Patent#7,531,572 Protocol

Animal Model for Assessing Therapeutic Efficacy of .alpha.-Amino Acid Prodrugs for Treating Anxiety. US Patent#7,531,572 Protocol

Animal Model for Assessing Therapeutic Efficacy of Prodrugs of 3-Aminopropylphosphinic Acid Analogs for Treating Depression. US Patent#7,585,996 Protocol

Animal Model for Assessing Therapeutic Efficacy of Prodrugs of 3-Aminopropylphosphinic Acid Analogs for Treating Alzheimer's Disease. US Patent#7,585,996 Protocol

Animal Model for Assessing Therapeutic Efficacy of Prodrugs of 3-Aminopropylphosphinic Acid Analogs for Treating Anxiety. US Patent#7,585,996 Protocol

Animal Model for Assessing Therapeutic Efficacy of Prodrugs of 3-Aminopropylphosphinic Acid Analogs for Treating Epilepsy. US Patent#7,585,996 Protocol

Animal Model of Nippostrongylus brasiliensis ... - Current Protocols Abstract Abstract: Animal models of Nippostrongylus brasiliensis and Heligmosomoides polygyrus infection are powerful tools for the investigation of the ... www.currentprotocols.com

Animal Models - Lab Protocol lab, laboratory, laboratory protocols, biology, life science, pcr, northern blot ... Animal Models, fsdf, 2009.07.05. Animal Models, fdsfdsfdxxx, 2009.07.05 ... www.labprotocol.com/index.php?kid=26...Animal+Models

Animal Models for Assessing Therapeutic Efficacy of .alpha.-Amino Acid Prodrugs for Treating Parkinson's Disease. US Patent#7,531,572 Protocol

Animal models for depression-like and - Nature.com 13 Dec 2007 ... Animal models for depression-like and anxiety-like behavior .... This protocol is related to the following articles: ... www.nature.com/protocolexchange/protocols/344

Animal Models for SLE | Current Protocols Abstract Abstract: Systemic lupus erythematosus (SLE) in humans is ... www.currentprotocols.com

Animal Models of Acute and Chronic Graft-Versus-Host Disease effective regimen in blocking T cell function in GVHD. Supplement 27. Current Protocols in Immunology. 4.3.4. Animal Models of. Acute and. Chronic ... onlinelibrary.wiley.com/doi/10.1002/0471142735.im0403s27/pdf

Animal Models of Depression. US Patent#7,531,572 Protocol

Animal Models of Pain. US Patent#7,531,572 Protocol

Animal Models of Painful Diabetic Neuropathy ... - Current Protocols Abstract Abstract: Painful peripheral neuropathy is a common secondary ... www.currentprotocols.com/protocol/ns0918

Animal Models of Retroviral Encephalopathies. 6 Jul 2009 ... Protocols, methods, and experimental design for pharmacology, drug development, drug screens, pharmacokinetics, ADME, preclinical models of ... www.scientistsolutions.com

Animal Models to Assess the Efficacy of .alpha.-Amino Acid Prodrugs for Treating Social Phobia. US Patent#7,531,572 Protocol

Anti-Hepatitis B Virus Effect of N-nonyl-DNJ Alone in a Woodchuck Model. US Patent#7,612,093 Protocol

Antiviral Study to Test the Activity of N-nonyl-DNJ in Combination with 3TC in a Woodchuck Model of Hepatitis B Virus Infection. US Patent#7,612,093 Protocol

Auditory Startle and Prepulse Inhibition of Startle (PPI) Animal Model of Schizophrenia. US Patent#7,531,572 Protocol

Chemically induced mouse models of intestinal inflammation. 15 Mar 2007 ... Animal models of intestinal inflammation are indispensable for our .... Additionally, a protocol to generate colitis-associated colorectal ... www.nature.com

Chronic Constriction Injury Model (CCI Model) US Patent#6,936,628 Protocol

Esophageal injury model in vivo tissue reconstitution. J Clin Invest. 2008 Dec;118(12):3860-9 Protocol

Establishment of rat liver transplantation model. Hepatobiliary Pancreat Dis Int. 2008 Feb;7(1):29-33 Protocol

Experimental Orthotopic Tumor Models. Proc Natl Acad Sci U S A. 2008 March 18; 105(11): 4329 4334 Protocol

Experimental protocols for CMD animal models Experimental protocols for CMD animal models. Raffaella Willmann. Raffaella Willmann studied Biology in Italy and obtained her PhD in Biochemistry in ... www.treat-nmd.eu/research/preclinical/cmd-sops/

Haloperidol-induced Catalepsy in the Rat. US Patent#7,572,802 Protocol

Hemiparkinsonian Rat Model. Stem Cells Vol. 26 No. 8 August 2008, pp. 2183 -2192 Protocol

Human Ovarian Cancer Animal Model. PLoS One. 2009; 4(10): e7670. Protocol

In Vivo Data Rat Penile Erection Model. US Patent#7,470,691 Protocol

In vivo model of airway inflammation. PLoS ONE 4(11): e7525. Protocol

In vivo model of airway inflammation. PLoS ONE 4(11): e7525. Protocol

Induction of PD in Rats. US Patent#6,914,056 Protocol

Lymphadema Animal Model. US Patent#7,476,384 Protocol

Method for Making Atopic Dermatitis Model Mice. US Patent Application#20090246181 Protocol

Method for Making Atopic Dermatitis Model Mice. US Patent Application#20090246181 Protocol

Middle cerebral artery occlusion (MCAO) model. Journal of Neuroscience, November 19, 2008, 28(47):12433-12444 Protocol

Mouse Model for Hepatic Repopulation. Stem Cells Vol. 26 No. 5 May 2008, pp. 1117 -1127 Protocol

Mouse Models of Periventricular Leukomalacia This video protocol demonstrate establishing mouse models of periventricular leukomalacia (PVL), the predominant form of brain injury in premature infants. http://www.jove.com/Details.php?ID=2074

Murine model of ascending Urinary Tract Infection. PLoS Pathog. 2009 September; 5(9): e1000586. Protocol

Murine Model of Hindlimb Ischemia This video demonstrates the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model are described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells. http://www.jove.com/details.stp?id=1035

Myocardial Infarction Model and Bulk Cell Transplantation. PLoS ONE. 2008; 3(3): e1789. Protocol

Osteoporosis Induction in Animal Model So, there was a need to have protocol in animal model to let the ... aim of this research was to make such protocol in animal model. ... www.scipub.org/fulltext/AJAV/AJAV52139-145.pdf

PCP-Induced Hyperactivity Animal Model of Schizophrenia. US Patent#7,531,572 Protocol

Production of a Parkinsonian Rat Model, Cell Transplantation, and Behavioral Testing. Proc Natl Acad Sci U S A. 2008 March 4; 105(9): 3392 3397 Protocol

Rat Cryoinjury Model. Proc Natl Acad Sci U S A. 2008 Apr 22;105(16):6063-8 Protocol

Rat model of chronic myocardial infarction. J Formos Med Assoc. 2008 Feb;107(2):165-74 Protocol

Rat model of myocardial infarction. J Cell Mol Med. 2008 Mar 28 Protocol

Rescue of Ischemia in Rabbit Lower Limb Model. US Patent#7,476,384 Protocol

Small Animal Models of Hemorrhagic Shock. 6 Jul 2009 ... Protocols and methods for immunology, antigen presentation ... www.scientistsolutions.com

Spinal Cord Injury Model and Transplantation. PLoS ONE 4(11): e7706. Protocol

Springer Protocols: Animal Models Animal Models. Protocols in Animal Models ... Fluorescent In Situ Hybridization Protocols in Drosophila Embryos and Tissues ... www.springerprotocols.com/.../browse?...Protocol...Animal%20Models...Animal%20Models

Stem ceII mobilisation- Mice model. WO/2008/104090 Protocol

Tissue Samples from a Murine Model of Induced Cardiovascular Defects. PLoS ONE. 2009; 4(1): e4221 Protocol

Tissue Samples from a Murine Model of Induced Cardiovascular Defects. PLoS ONE. 2009; 4(1): e4221 Protocol

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice A protocol to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. http://www.jove.com/index/details.stp?ID=404