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 β 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.
Similar content being viewed by others
Author information
Authors and Affiliations
Additional information
Accepted: 30 June 1999
Rights and permissions
About this article
Cite this article
Tullberg-Reinert, H., Jundt, G. In situ measurement of collagen synthesis by human bone cells with a Sirius Red-based colorimetric microassay: effects of transforming growth factor β2 and ascorbic acid 2-phosphate. Histochemistry 112, 271–276 (1999). https://doi.org/10.1007/s004180050447
Issue Date:
DOI: https://doi.org/10.1007/s004180050447