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.
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.
Tullberg-Reinert
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H
Laboratory of Experimental Osteology, Institute of Pathology, Kantonsspital/University Clinics, Schonbeinstrasse 40, CH-4003 Basle, Switzerland. htullberg@uhbs.ch
Jundt
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D000293:Adolescent; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001391:Azo Compounds / Q000032:analysis; D003094:Collagen / Q000032:analysis / Q000096:biosynthesis*; D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents / Q000032:analysis*; D003907:Dexamethasone / Q000494:pharmacology; D004347:Drug Interactions; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D001391:Azo Compounds; D004396:Coloring Agents; D016212:Transforming Growth Factor beta; C009798:C.I. direct red 80; C011669:ascorbate-2-phosphate; D003907:Dexamethasone; D003094:Collagen; D001205:Ascorbic Acid