Fibrin In Vitro Angiogenesis A

Fibrin In Vitro Angiogenesis Assay

Chemicon (Millipore)

Introduction

Angiogenesis is the process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth and metastasis)1.

The CHEMICON Fibrin Gel In Vitro Angiogenesis Assay Kit provides a convenient system for evaluation of tube formation by endothelial cells in 96-well or other formats. When cultured on top or within a fibrin gel, endothelial cells rapidly align and form interconnecting networks that can display patent lumina2,3. Tube formation is a multi-step process involving cell adhesion, migration, differentiation and growth4. The formation of intercellular connections and lumina within endothelial cell networks in fibrin gels is dependent upon the actions of VE-cadherin, avb3 and a5b1 integrins, the cdc42 and Rac1 GTPases, and membrane-type matrix metalloproteinases (MT-MMPs)2,3,5,6. Angiogenesis within fibrin gels in vitro is regarded as an accurate model for wound healing and tumor angiogenesis, as tumor cell-derived vascular endothelial growth factor/vascular permeability factor promotes leakage of fibrinogen from the tumor vasculature and formation of a fibrin-rich proangiogenic provisional matrix7.

Fibrin gel formation is initiated by enzymatic cleavage of fibrinogen, a heterotrimer, by thrombin8. The resulting cleaved fibrin molecules form regular, multimolecular arrays that are highly translucent. The concentrations and formulations of the fibrinogen and thrombin in this kit are optimized for maximal tube-formation by HUVEC and easy visualization of these tubes.

Activity Assay

Application

The CHEMICON Fibrin Gel In Vitro Angiogenesis Assay Kit represents a simple model of angiogenesis in which the induction or inhibition of tube formation by exogenous signals can be easily monitored. Fibrin gels are easily and quickly formed in culture dishes by mixing Fibrinogen and Thrombin Solutions. For assaying inhibitors or stimulators of tube formation, simply premix the endothelial cell suspension with different concentrations of the inhibitor or stimulator to be tested, before adding the cells to the top of the fibrin gel. Addition of a second layer of fibrin gel on the day after plating the cells is optional, but will promote optimal survival and a higher degree of network and lumen formation. The assay can be used to monitor the extent of tube assembly in various endothelial cells, e.g. human umbilical vein cells (HUVEC) or bovine capillary endothelial (BCE) cells.

Vertebrates

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Cell Based Assays

• Fibrinogen Solution: (Part No. 90244) One 10 mL bottle.
• Thrombin solution: (Part No. 90246) One 7.5 mL bottle.
• 100x Positive Control Angiogenic Supplement: (Part No. 90247) One 0.5 mL vial. (Contains1.0 mg/ml insulin from bovine pancreas, 0.55 mg/ml human transferrin (substantially iron-free), and 0.5 μg/ml sodium selenite in EBSS without phenol red).

Store unopened kit materials at -20°C or -70°C for up to their expiration date. Once Fibrinogen Solution has been thawed, store at room temperature for up to two weeks. Thawed Thrombin Solution should be stored at 4ºC for up to one month. Thawed 100x Positive Control Angiogenic Supplement can be stored at 4ºC for up to one year.

1. 96-well or other sized Tissue Culture plate

2. Microcentrifuge Tubes, sterile

3. 37°C Tissue Culture Incubator

1. Inverted Light Microscope

2. HUVEC cells or other experimental cell line (any species), and appropriate growth medium and subculturing reagents. Early passage cells are preferred, as they are less prone to cell death in this assay.

3. Pipet capable of delivering 100-200 μL, preferably a multichannel pipet with 96-well plate

4. Basal medium - EBM (Cambrex/BioWhittaker) or MCDB131 (Sigma) supplemented with 1% BSA and antibiotic.

5. PMA (optional) to add to basal media and angiogenic supplement for positive control.