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Histochem Cell Biol. 1999 Oct;112(4):271-6.

In situ measurement of collagen synthesis by human bone cells with a sirius red-based colorimetric microassay: effects of transforming growth factor beta2 and ascorbic acid 2-phosphate.

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  • 1Laboratory of Experimental Osteology, Institute of Pathology, Kantonsspital/University Clinics, Schonbeinstrasse 40, CH-4003 Basle, Switzerland. htullberg@uhbs.ch

Abstract

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid beta were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples.

PMID:
10550611
[PubMed - indexed for MEDLINE]
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