In the June 2009 issue
Collagen Type II for in vitro and in vivo applications
In Vitro Applications in Inflammation
Collagen Type II for in vitro and in vivo applications.
In vitro use:
Collagen Type II coated microplates for cell attachment/inhibition assays
Extracellular matrix is primarily made up of two major components—proteoglycans and collagens—with collagen type II (COL2a, CII) being the predominant collagen in cartilage. CII, along with other smaller collagens, forms a three-dimensional fibrillar network essential for the tensile strength of cartilage. Aggrecans and other proteoglycans are embedded in this fibrous network, providing compressibility and elasticity.
Chondrocytes are the cells responsible for the synthesis, organization and
maintenance of the ECM. Normal matrix turnover is dependant on the ability of chondrocytes to detect alterations in the ECM. To date, there are still many unknowns in the chondrocyte physiology. Common assays for studying chondrocyte biology are cell attachment and inhibition assays using microplates coated with typeII collagen, which encourages chondrocyte proliferation and differentiation. MD Biosciences carries a line of Collagen Type II for the study of
chondrocytes.
In vivo use:
Collagen Type II for induction of RA-like disease in the Collagen-Induced Arthritis (CIA) model
Collagen
Type II, along with adjuvant, is used in a common model of Collagen-Induced Arthritis for the study of Rheumatoid Arthritis. The model is widely used, and the disease severity and incidence can vary greatly between laboratories. The quality of Type II Collagen, along with other factors, can reduce the disease incidence/severity. MD Biosciences' collagen type II is >99 % purity, contributing to higher incidence/severity.
Ways to increase incidence/severity in the Collagen-Induced Arthritis model:
- Collagen: only use a highly-purified source of collagen (>99.9%). Ensure that the collagen has not denatured. Keep collagen cold and stored appropriately. Collagen should be a clear solution. If cloudy, the collagen may have cross-linked and precipitated, or it has been
contaminated.
- Unstable emulsion: the preparation of the CII and CFA emulsion is a critical step. Insufficient mixing and temperature are common errors at this step. Increase the speed of the homogenizer (or other mixing apparatus), and keep the reagents and mixing vessel on ice during the emulsification step.
- Animal health, age, strain: Mice around 8 weeks of age are recommended for the CIA model. Male DBA/1 mice 8 weeks of age are recommended for the CIA model. The estrogen in female mice may effect the severity/incidence of disease. If other strains such as the C57Bl/6 mouse are preferred, CII from chicken would be recommended over bovine sources.
| Collagen |
Qty |
Catalog # |
| Bovine Collagen Type II |
10 mg |
804001 |
| Chicken Collagen Type II |
10 mg |
804002 |
| Rat pepsin-digested Type II Collagen |
0.5 mg |
8041002 |
| Rat Lathyrtic Type II Collagen |
0.5 mg |
8041001 |
In addition to offering Collagens for disease induction, MD Biosciences is a preclinical contract research organization (CRO) offering efficacy studies for Collagen Induced Arthritis (CIA) and Collagen Antibody Induced Arthritis (CAIA)
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Applications in Inflammation: In vitro screening, compound profiling and mechanism of action.
The immune system plays an important role in the pathogenesis of many autoimmune conditions such as asthma/allergies, rheumatoid arthritis and multiple sclerosis as well as diseases such as IBD, cancer, artherosclerosis and infectious diseases. An understanding of the cellular processes involved and how proposed therapies modulate the immune response pathways provides important information during the drug discovery process.
The immune system is capable of mounting two types of responses: the innate immune response, a rapid and relatively non-selective response that controls infection, and the adaptive immune response, a highly specific response with T and B cells as its main components. The innate response is comprised of inflammatory cells as well as other cells such as dendritic cells, which communicate with the adaptive immune response. While the two immune responses working together is a desired effect during an infection or insult, an out of control immune response can lead to autoimmune conditions. Many biopharmaceutical companies are focusing on immunomodulatory drugs that can selectively target immune cell types and the innate immune response, which can then trigger the appropriate adaptive immune response.
Determining the Molecular Mode of Action Using In Vitro Assays
Extracellular cues are transmitted through the cell by a network of signal transduction molecules. A compound's mode of action can be further characterized by identifying pathways affected in the following ways:
- Identify inhibitors or agonists of cell signaling pathways by determining the phosphorylation state of intracellular proteins such as Akt, CREB, ERK1/2, GSK-3ß, HSP27, IkBa, JNK, p38 MAPK, p70S6K and ZAP-70. Concentrate on a specific pathway, such as T cell activation, or screen multiple pathways at once.
- Screen compounds for teir ability to activate or inhibit a specific pathway using cell
lines harboring a luciferase reporter gene under the control of NFkB, STAT-1, STAT-3, AP-1, CREB or NFAT responsive elements.
- Determine second messenger levels, such cAMP or calcium, in compound treated cells.
- Screen compounds for their ability to inhibit enzyme activity: Kinase activity, cyclooxygenase activity (COX-1, COX-2), monoamine oxidase activity (MAO), aggrecanase activity, matrix metalloproteinase activity.
MD Biosciences In Vitro Discovery Group can work with your team to establish a custom screening program to determine the mechanism of action of your compound. Contact us for a customized approach tailored to your compound class.
In Vitro Assay:
Inflammatory Related Assays
Cell Signaling Pathway Screens
Enzyme Inhibition Assays
Calcium Flux Assays
Adaptive Immune Response Assays