Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity
Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variabil...
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| Veröffentlicht in: | Journal of immunological methods 1998-02, Vol.211 (1), p.159-169 |
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| container_title | Journal of immunological methods |
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| creator | Blaheta, Roman A. Kronenberger, Bernd Woitaschek, Dirk Weber, Stephan Scholz, Martin Schuldes, Horst Encke, Albrecht Markus, Bernd H. |
| description | Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen™. PicoGreen™ has been shown to detect as little as 0.5 ng pure DNA or 10
2 cells (interassay SD |
| doi_str_mv | 10.1016/S0022-1759(97)00202-0 |
| format | Article |
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2 cells (interassay SD<10%, intraassay SD<5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen™, cells were digested with papain for 20 h at 60°C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen™, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen™. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/S0022-1759(97)00202-0</identifier><identifier>PMID: 9617840</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Animals ; Biological and medical sciences ; Calibration ; Cattle ; Cell cycle, cell proliferation ; Cell Division ; Cell number quantification ; Cell physiology ; Cells, Cultured ; DNA - analysis ; Endothelium, Vascular ; Fluorescence assay ; Fluorescent Dyes ; Fundamental and applied biological sciences. Psychology ; Hoechst 33342 ; Humans ; Liver - cytology ; Mitosis - physiology ; Molecular and cellular biology ; Organic Chemicals ; Papain ; PicoGreen ; Primary cell cultures ; Propidium iodide ; Rats ; Sensitivity and Specificity</subject><ispartof>Journal of immunological methods, 1998-02, Vol.211 (1), p.159-169</ispartof><rights>1998 Elsevier Science B.V.</rights><rights>1998 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c472t-9f35f68a9be2aa807e1f9321a83c9879bd4a0643b04a58f8cc9a852bc44dc14b3</citedby><cites>FETCH-LOGICAL-c472t-9f35f68a9be2aa807e1f9321a83c9879bd4a0643b04a58f8cc9a852bc44dc14b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0022-1759(97)00202-0$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=2243091$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9617840$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Blaheta, Roman A.</creatorcontrib><creatorcontrib>Kronenberger, Bernd</creatorcontrib><creatorcontrib>Woitaschek, Dirk</creatorcontrib><creatorcontrib>Weber, Stephan</creatorcontrib><creatorcontrib>Scholz, Martin</creatorcontrib><creatorcontrib>Schuldes, Horst</creatorcontrib><creatorcontrib>Encke, Albrecht</creatorcontrib><creatorcontrib>Markus, Bernd H.</creatorcontrib><title>Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen™. PicoGreen™ has been shown to detect as little as 0.5 ng pure DNA or 10
2 cells (interassay SD<10%, intraassay SD<5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen™, cells were digested with papain for 20 h at 60°C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen™, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen™. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Calibration</subject><subject>Cattle</subject><subject>Cell cycle, cell proliferation</subject><subject>Cell Division</subject><subject>Cell number quantification</subject><subject>Cell physiology</subject><subject>Cells, Cultured</subject><subject>DNA - analysis</subject><subject>Endothelium, Vascular</subject><subject>Fluorescence assay</subject><subject>Fluorescent Dyes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hoechst 33342</subject><subject>Humans</subject><subject>Liver - cytology</subject><subject>Mitosis - physiology</subject><subject>Molecular and cellular biology</subject><subject>Organic Chemicals</subject><subject>Papain</subject><subject>PicoGreen</subject><subject>Primary cell cultures</subject><subject>Propidium iodide</subject><subject>Rats</subject><subject>Sensitivity and Specificity</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhS0EKkPhJ1TyAiFYBK4dJ7ZXqCpPqRILYG05zg0YJfFgO1Pm3-N0otl2ZVn3O-c-DiFXDN4yYO277wCcV0w2-rWWb8oHeAWPyI4pySupoXlMdmfkKXmW0h8AYNDCBbnQLZNKwI78-4AHHMN-wjnTMFA702XM0Sack8_-gNTP9OBzDNSmZI80BzqF2ecQ6a8Y7vLvVbWPfrLxSB2OI3XFYImY6J0v1Yj94rCnU5Fk76h1xdXn43PyZLBjwhfbe0l-fvr44-ZLdfvt89eb69vKCclzpYe6GVpldYfcWgUS2aBrzqyqnVZSd72w0Iq6A2EbNSjntFUN75wQvWOiqy_Jq5PvPoa_C6ZsJp_WOe2MYUlGas1aoeFBkLUc6rZWBWxOoIshpYiD2dY3DMwajbmPxqx3N1qa-2jM2uBqa7B0E_Zn1ZZFqb_c6jY5Ow7Rzs6nM8a5qEGzgr0_YViudvAYTXIe53JjH9Fl0wf_wCD_Ad1frPU</recordid><startdate>19980201</startdate><enddate>19980201</enddate><creator>Blaheta, Roman A.</creator><creator>Kronenberger, Bernd</creator><creator>Woitaschek, Dirk</creator><creator>Weber, Stephan</creator><creator>Scholz, Martin</creator><creator>Schuldes, Horst</creator><creator>Encke, Albrecht</creator><creator>Markus, Bernd H.</creator><general>Elsevier B.V</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>7QO</scope><scope>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19980201</creationdate><title>Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity</title><author>Blaheta, Roman A. ; Kronenberger, Bernd ; Woitaschek, Dirk ; Weber, Stephan ; Scholz, Martin ; Schuldes, Horst ; Encke, Albrecht ; Markus, Bernd H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c472t-9f35f68a9be2aa807e1f9321a83c9879bd4a0643b04a58f8cc9a852bc44dc14b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Calibration</topic><topic>Cattle</topic><topic>Cell cycle, cell proliferation</topic><topic>Cell Division</topic><topic>Cell number quantification</topic><topic>Cell physiology</topic><topic>Cells, Cultured</topic><topic>DNA - analysis</topic><topic>Endothelium, Vascular</topic><topic>Fluorescence assay</topic><topic>Fluorescent Dyes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hoechst 33342</topic><topic>Humans</topic><topic>Liver - cytology</topic><topic>Mitosis - physiology</topic><topic>Molecular and cellular biology</topic><topic>Organic Chemicals</topic><topic>Papain</topic><topic>PicoGreen</topic><topic>Primary cell cultures</topic><topic>Propidium iodide</topic><topic>Rats</topic><topic>Sensitivity and Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Blaheta, Roman A.</creatorcontrib><creatorcontrib>Kronenberger, Bernd</creatorcontrib><creatorcontrib>Woitaschek, Dirk</creatorcontrib><creatorcontrib>Weber, Stephan</creatorcontrib><creatorcontrib>Scholz, Martin</creatorcontrib><creatorcontrib>Schuldes, Horst</creatorcontrib><creatorcontrib>Encke, Albrecht</creatorcontrib><creatorcontrib>Markus, Bernd H.</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>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Blaheta, Roman A.</au><au>Kronenberger, Bernd</au><au>Woitaschek, Dirk</au><au>Weber, Stephan</au><au>Scholz, Martin</au><au>Schuldes, Horst</au><au>Encke, Albrecht</au><au>Markus, Bernd H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>1998-02-01</date><risdate>1998</risdate><volume>211</volume><issue>1</issue><spage>159</spage><epage>169</epage><pages>159-169</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity. Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability. For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen™. PicoGreen™ has been shown to detect as little as 0.5 ng pure DNA or 10
2 cells (interassay SD<10%, intraassay SD<5%). This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide. To obtain optimum efficacy of PicoGreen™, cells were digested with papain for 20 h at 60°C prior to staining. Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell). Analysis of the blank values revealed a very low autofluorescence of PicoGreen™, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide. Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen™. In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>9617840</pmid><doi>10.1016/S0022-1759(97)00202-0</doi><tpages>11</tpages></addata></record> |
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| ispartof | Journal of immunological methods, 1998-02, Vol.211 (1), p.159-169 |
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| subjects | Animals Biological and medical sciences Calibration Cattle Cell cycle, cell proliferation Cell Division Cell number quantification Cell physiology Cells, Cultured DNA - analysis Endothelium, Vascular Fluorescence assay Fluorescent Dyes Fundamental and applied biological sciences. Psychology Hoechst 33342 Humans Liver - cytology Mitosis - physiology Molecular and cellular biology Organic Chemicals Papain PicoGreen Primary cell cultures Propidium iodide Rats Sensitivity and Specificity |
| title | Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity |
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