A microassay to quantitate collagen synthesis by cells in culture
A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [ 3H]proline. After incubation, the plates were heated to 90°C to s...
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| Veröffentlicht in: | Analytical biochemistry 1990-05, Vol.186 (2), p.296-300 |
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| creator | Diegelmann, R.F. Bryson, G.R. Flood, L.C. Graham, M.F. |
| description | A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [
3H]proline. After incubation, the plates were heated to 90°C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble
3H-labeled collagen-derived peptides and the remaining insoluble
3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and noncollagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material. |
| doi_str_mv | 10.1016/0003-2697(90)90083-L |
| format | Article |
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3H]proline. After incubation, the plates were heated to 90°C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble
3H-labeled collagen-derived peptides and the remaining insoluble
3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and noncollagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(90)90083-L</identifier><identifier>PMID: 2163587</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Cells, Cultured ; Collagen - biosynthesis ; DNA - biosynthesis ; fibroblasts ; Fibroblasts - metabolism ; Fundamental and applied biological sciences. Psychology ; Glycoproteins ; Humans ; intestine ; Intestines - cytology ; Microbial Collagenase ; Microchemistry ; Muscle, Smooth - cytology ; Muscle, Smooth - metabolism ; Proline - analysis ; Protein Biosynthesis ; Proteins ; Skin ; smooth muscle</subject><ispartof>Analytical biochemistry, 1990-05, Vol.186 (2), p.296-300</ispartof><rights>1990</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c417t-bd2cef862fb69645e6c98892ce32c305458ccb9137e8644474dffc1a7c11b9713</citedby><cites>FETCH-LOGICAL-c417t-bd2cef862fb69645e6c98892ce32c305458ccb9137e8644474dffc1a7c11b9713</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/000326979090083L$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6934311$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2163587$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Diegelmann, R.F.</creatorcontrib><creatorcontrib>Bryson, G.R.</creatorcontrib><creatorcontrib>Flood, L.C.</creatorcontrib><creatorcontrib>Graham, M.F.</creatorcontrib><title>A microassay to quantitate collagen synthesis by cells in culture</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [
3H]proline. After incubation, the plates were heated to 90°C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble
3H-labeled collagen-derived peptides and the remaining insoluble
3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and noncollagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Cells, Cultured</subject><subject>Collagen - biosynthesis</subject><subject>DNA - biosynthesis</subject><subject>fibroblasts</subject><subject>Fibroblasts - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glycoproteins</subject><subject>Humans</subject><subject>intestine</subject><subject>Intestines - cytology</subject><subject>Microbial Collagenase</subject><subject>Microchemistry</subject><subject>Muscle, Smooth - cytology</subject><subject>Muscle, Smooth - metabolism</subject><subject>Proline - analysis</subject><subject>Protein Biosynthesis</subject><subject>Proteins</subject><subject>Skin</subject><subject>smooth muscle</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEtv1DAQgC0EKkvhH4DkA6rgkGLHjh8XpFXVFqSVuJSz5UwmYJRNWo-DtP-ehF3tEU4jzXzz-hh7K8W1FNJ8EkKoqjbefvDioxfCqWr3jG2k8KYSSvjnbHNGXrJXRL-EkFI35oJd1NKoxtkN2275PkGeIlE88DLxpzmOJZVYkMM0DPEHjpwOY_mJlIi3Bw44DMTTyGEeypzxNXvRx4HwzSlesu93tw83X6rdt_uvN9tdBVraUrVdDdg7U_et8UY3aMA755ekqkGJRjcOoPVSWXRGa2111_cgowUpW2-lumRXx7mPeXqakUrYJ1qPiSNOMwXrnXba1v8FZbPsFY1ZQH0El_-JMvbhMad9zIcgRVgVh9VfWP0FL8JfxWG3tL07zZ_bPXbnppPTpf7-VI8EcehzHCHRGTNeaSXXfz4fMVyk_U6YA0HCEbBLGaGEbkr_vuMPnuyWxQ</recordid><startdate>19900501</startdate><enddate>19900501</enddate><creator>Diegelmann, R.F.</creator><creator>Bryson, G.R.</creator><creator>Flood, L.C.</creator><creator>Graham, M.F.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19900501</creationdate><title>A microassay to quantitate collagen synthesis by cells in culture</title><author>Diegelmann, R.F. ; Bryson, G.R. ; Flood, L.C. ; Graham, M.F.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c417t-bd2cef862fb69645e6c98892ce32c305458ccb9137e8644474dffc1a7c11b9713</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Cells, Cultured</topic><topic>Collagen - biosynthesis</topic><topic>DNA - biosynthesis</topic><topic>fibroblasts</topic><topic>Fibroblasts - metabolism</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glycoproteins</topic><topic>Humans</topic><topic>intestine</topic><topic>Intestines - cytology</topic><topic>Microbial Collagenase</topic><topic>Microchemistry</topic><topic>Muscle, Smooth - cytology</topic><topic>Muscle, Smooth - metabolism</topic><topic>Proline - analysis</topic><topic>Protein Biosynthesis</topic><topic>Proteins</topic><topic>Skin</topic><topic>smooth muscle</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Diegelmann, R.F.</creatorcontrib><creatorcontrib>Bryson, G.R.</creatorcontrib><creatorcontrib>Flood, L.C.</creatorcontrib><creatorcontrib>Graham, M.F.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Diegelmann, R.F.</au><au>Bryson, G.R.</au><au>Flood, L.C.</au><au>Graham, M.F.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A microassay to quantitate collagen synthesis by cells in culture</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1990-05-01</date><risdate>1990</risdate><volume>186</volume><issue>2</issue><spage>296</spage><epage>300</epage><pages>296-300</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [
3H]proline. After incubation, the plates were heated to 90°C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble
3H-labeled collagen-derived peptides and the remaining insoluble
3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and noncollagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>2163587</pmid><doi>10.1016/0003-2697(90)90083-L</doi><tpages>5</tpages></addata></record> |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Cells, Cultured Collagen - biosynthesis DNA - biosynthesis fibroblasts Fibroblasts - metabolism Fundamental and applied biological sciences. Psychology Glycoproteins Humans intestine Intestines - cytology Microbial Collagenase Microchemistry Muscle, Smooth - cytology Muscle, Smooth - metabolism Proline - analysis Protein Biosynthesis Proteins Skin smooth muscle |
| title | A microassay to quantitate collagen synthesis by cells in culture |
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