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A quantitative ELISA‐based co‐culture angiogenesis and cell proliferation assay
Author(s) -
FRIIS TINA,
KJÆR SØRENSEN BIRGITTE,
ENGEL ANNEMARIE,
RYGAARD JØRGEN,
HOUEN GUNNAR
Publication year - 2003
Publication title -
apmis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.909
H-Index - 88
eISSN - 1600-0463
pISSN - 0903-4641
DOI - 10.1034/j.1600-0463.2003.1110609.x
Subject(s) - angiogenesis , umbilical vein , human umbilical vein endothelial cell , endothelial stem cell , angiogenesis inhibitor , chemistry , cell culture , basic fibroblast growth factor , fibroblast growth factor , cell growth , microbiology and biotechnology , biology , cancer research , biochemistry , growth factor , in vitro , genetics , receptor
Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well‐tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335–44) to a quantitative ELISA‐based fibroblast and endothelial cell co‐culture angiogenesis assay. In each well of a 96‐microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co‐culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well‐known angiogenesis inhibitors on HUVEC tube formation.