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An Improved Method for Determining Proteoglycans Synthesized by Chondrocytes in Culture
| Content Provider | Scilit |
|---|---|
| Author | Goldberg, Ronald L. Kolibas, Lorraine M. |
| Copyright Year | 1990 |
| Description | An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes. |
| Related Links | http://www.tandfonline.com/doi/pdf/10.3109/03008209009152154 |
| Ending Page | 275 |
| Page Count | 11 |
| Starting Page | 265 |
| ISSN | 03008207 |
| e-ISSN | 16078438 |
| DOI | 10.3109/03008209009152154 |
| Journal | Connective tissue research |
| Issue Number | 3-4 |
| Volume Number | 24 |
| Language | English |
| Publisher | Informa UK Limited |
| Publisher Date | 1990-01-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Connective Tissue Research Biochemical Research Glycosaminoglycans Metachromasia |
| Content Type | Text |
| Resource Type | Article |
| Subject | Orthopedics and Sports Medicine Cell Biology Molecular Biology Biochemistry Rheumatology |
