A rapid method to quantify neurons in mixed cultures based on the specific binding of [ 3H]ouabain to neuronal Na +,K +-ATPase
The high-affinity binding of [ 3H]ouabain to Na +,K +-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [ 3H]ouabain was found to be time-dependent, high-affinity (apparent K d= 8.5nM), saturable ( B max= 20.6pmol/mg pro...
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| Veröffentlicht in: | Brain research 1991-01, Vol.538 (1), p.1-8 |
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| creator | Markwell, M.A.K. Sheng, H.Z. Brenneman, D.E. Paul, S.M. |
| description | The high-affinity binding of [
3H]ouabain to Na
+,K
+-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [
3H]ouabain was found to be time-dependent, high-affinity (apparent
K
d= 8.5nM), saturable (
B
max= 20.6pmol/mg protein), dependent upon the presence of ATP, inhibited by K
+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [
3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins
N-methyl-
d-aspartate (NMDA) and kainate, reduced the amount of [
3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [
3H]ouabain binding to the neuronal form of Na
+,K
+-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture. |
| doi_str_mv | 10.1016/0006-8993(91)90368-6 |
| format | Article |
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3H]ouabain to Na
+,K
+-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [
3H]ouabain was found to be time-dependent, high-affinity (apparent
K
d= 8.5nM), saturable (
B
max= 20.6pmol/mg protein), dependent upon the presence of ATP, inhibited by K
+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [
3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins
N-methyl-
d-aspartate (NMDA) and kainate, reduced the amount of [
3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [
3H]ouabain binding to the neuronal form of Na
+,K
+-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.</description><identifier>ISSN: 0006-8993</identifier><identifier>EISSN: 1872-6240</identifier><identifier>DOI: 10.1016/0006-8993(91)90368-6</identifier><identifier>PMID: 1850316</identifier><identifier>CODEN: BRREAP</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Biological and medical sciences ; Cell Survival - drug effects ; Cells, Cultured ; Culture Techniques - methods ; Fundamental and applied biological sciences. Psychology ; General aspects. Models. Methods ; Hippocampus ; Hippocampus - cytology ; Kainate ; Kainic Acid - pharmacology ; Kinetics ; N-Methyl- d-aspartate ; N-Methylaspartate - pharmacology ; Na +,K +-ATPase ; Neurons - cytology ; Neurons - drug effects ; Neurons - enzymology ; Neurotoxicity ; Ouabain ; Ouabain - metabolism ; Phencyclidine ; Protein Binding ; Radioisotope Dilution Technique ; Sodium-Potassium-Exchanging ATPase - metabolism ; Tritium ; Vertebrates: nervous system and sense organs</subject><ispartof>Brain research, 1991-01, Vol.538 (1), p.1-8</ispartof><rights>1991 Elsevier Science Publishers B.V. (Biomedical Division)</rights><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c387t-2a63c24deb8cf8937e720b0185483108342164de87eb0767dfb907c897f9c8513</citedby><cites>FETCH-LOGICAL-c387t-2a63c24deb8cf8937e720b0185483108342164de87eb0767dfb907c897f9c8513</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0006-8993(91)90368-6$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19436586$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1850316$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Markwell, M.A.K.</creatorcontrib><creatorcontrib>Sheng, H.Z.</creatorcontrib><creatorcontrib>Brenneman, D.E.</creatorcontrib><creatorcontrib>Paul, S.M.</creatorcontrib><title>A rapid method to quantify neurons in mixed cultures based on the specific binding of [ 3H]ouabain to neuronal Na +,K +-ATPase</title><title>Brain research</title><addtitle>Brain Res</addtitle><description>The high-affinity binding of [
3H]ouabain to Na
+,K
+-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [
3H]ouabain was found to be time-dependent, high-affinity (apparent
K
d= 8.5nM), saturable (
B
max= 20.6pmol/mg protein), dependent upon the presence of ATP, inhibited by K
+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [
3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins
N-methyl-
d-aspartate (NMDA) and kainate, reduced the amount of [
3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [
3H]ouabain binding to the neuronal form of Na
+,K
+-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.</description><subject>Biological and medical sciences</subject><subject>Cell Survival - drug effects</subject><subject>Cells, Cultured</subject><subject>Culture Techniques - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>General aspects. Models. Methods</subject><subject>Hippocampus</subject><subject>Hippocampus - cytology</subject><subject>Kainate</subject><subject>Kainic Acid - pharmacology</subject><subject>Kinetics</subject><subject>N-Methyl- d-aspartate</subject><subject>N-Methylaspartate - pharmacology</subject><subject>Na +,K +-ATPase</subject><subject>Neurons - cytology</subject><subject>Neurons - drug effects</subject><subject>Neurons - enzymology</subject><subject>Neurotoxicity</subject><subject>Ouabain</subject><subject>Ouabain - metabolism</subject><subject>Phencyclidine</subject><subject>Protein Binding</subject><subject>Radioisotope Dilution Technique</subject><subject>Sodium-Potassium-Exchanging ATPase - metabolism</subject><subject>Tritium</subject><subject>Vertebrates: nervous system and sense organs</subject><issn>0006-8993</issn><issn>1872-6240</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1rFTEUhoMo9Vr9BwrZWJQ6mkzm5mMjXIq1xaIu6kokZJITG5lJbpOZYjf-dnM7F7tzFQ7neV9yHoSeU_KWEsrfEUJ4I5VirxR9rQjjsuEP0IpK0Ta87chDtPqHPEZPSvlVR8YUOUAHVK4Jo3yF_mxwNtvg8AjTVXJ4Svh6NnEK_hZHmHOKBYeIx_AbHLbzMM0ZCu5NqWOKeLoCXLZggw8W9yG6EH_i5PF3zM5-pNn0poZr51JlBvzZ4OM3n_Bxs7n8WkueokfeDAWe7d9D9O30w-XJWXPx5eP5yeaisUyKqWkNZ7btHPTSeqmYANGSntQrOskokaxrKa9rKaAnggvne0WElUp4ZeWaskN0tPRuc7qeoUx6DMXCMJgIaS5aknXLZKsq2C2gzamUDF5vcxhNvtWU6J12vXOqd061ovpOu-Y19mLfP_cjuPvQ4rnuX-73plgz-GyiDeUeUx3ja7nj3i8cVBk3AbIuNkC04EIGO2mXwv8_8hdSKZy1</recordid><startdate>19910104</startdate><enddate>19910104</enddate><creator>Markwell, M.A.K.</creator><creator>Sheng, H.Z.</creator><creator>Brenneman, D.E.</creator><creator>Paul, S.M.</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>7X8</scope></search><sort><creationdate>19910104</creationdate><title>A rapid method to quantify neurons in mixed cultures based on the specific binding of [ 3H]ouabain to neuronal Na +,K +-ATPase</title><author>Markwell, M.A.K. ; Sheng, H.Z. ; Brenneman, D.E. ; Paul, S.M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c387t-2a63c24deb8cf8937e720b0185483108342164de87eb0767dfb907c897f9c8513</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>Biological and medical sciences</topic><topic>Cell Survival - drug effects</topic><topic>Cells, Cultured</topic><topic>Culture Techniques - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>General aspects. Models. Methods</topic><topic>Hippocampus</topic><topic>Hippocampus - cytology</topic><topic>Kainate</topic><topic>Kainic Acid - pharmacology</topic><topic>Kinetics</topic><topic>N-Methyl- d-aspartate</topic><topic>N-Methylaspartate - pharmacology</topic><topic>Na +,K +-ATPase</topic><topic>Neurons - cytology</topic><topic>Neurons - drug effects</topic><topic>Neurons - enzymology</topic><topic>Neurotoxicity</topic><topic>Ouabain</topic><topic>Ouabain - metabolism</topic><topic>Phencyclidine</topic><topic>Protein Binding</topic><topic>Radioisotope Dilution Technique</topic><topic>Sodium-Potassium-Exchanging ATPase - metabolism</topic><topic>Tritium</topic><topic>Vertebrates: nervous system and sense organs</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Markwell, M.A.K.</creatorcontrib><creatorcontrib>Sheng, H.Z.</creatorcontrib><creatorcontrib>Brenneman, D.E.</creatorcontrib><creatorcontrib>Paul, S.M.</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>MEDLINE - Academic</collection><jtitle>Brain research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Markwell, M.A.K.</au><au>Sheng, H.Z.</au><au>Brenneman, D.E.</au><au>Paul, S.M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A rapid method to quantify neurons in mixed cultures based on the specific binding of [ 3H]ouabain to neuronal Na +,K +-ATPase</atitle><jtitle>Brain research</jtitle><addtitle>Brain Res</addtitle><date>1991-01-04</date><risdate>1991</risdate><volume>538</volume><issue>1</issue><spage>1</spage><epage>8</epage><pages>1-8</pages><issn>0006-8993</issn><eissn>1872-6240</eissn><coden>BRREAP</coden><abstract>The high-affinity binding of [
3H]ouabain to Na
+,K
+-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes. The specific binding of [
3H]ouabain was found to be time-dependent, high-affinity (apparent
K
d= 8.5nM), saturable (
B
max= 20.6pmol/mg protein), dependent upon the presence of ATP, inhibited by K
+, and directly proportional to neuronal, but not glial, cell number. Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture. At the concentration of neurons routinely used, the specific binding of [
3H]ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding. Agents that selectively kill neurons rather than glia, such as the excitotoxins
N-methyl-
d-aspartate (NMDA) and kainate, reduced the amount of [
3H]ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner. Measurement of high-affinity [
3H]ouabain binding to the neuronal form of Na
+,K
+-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>1850316</pmid><doi>10.1016/0006-8993(91)90368-6</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
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| ispartof | Brain research, 1991-01, Vol.538 (1), p.1-8 |
| issn | 0006-8993 1872-6240 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_80523829 |
| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Biological and medical sciences Cell Survival - drug effects Cells, Cultured Culture Techniques - methods Fundamental and applied biological sciences. Psychology General aspects. Models. Methods Hippocampus Hippocampus - cytology Kainate Kainic Acid - pharmacology Kinetics N-Methyl- d-aspartate N-Methylaspartate - pharmacology Na +,K +-ATPase Neurons - cytology Neurons - drug effects Neurons - enzymology Neurotoxicity Ouabain Ouabain - metabolism Phencyclidine Protein Binding Radioisotope Dilution Technique Sodium-Potassium-Exchanging ATPase - metabolism Tritium Vertebrates: nervous system and sense organs |
| title | A rapid method to quantify neurons in mixed cultures based on the specific binding of [ 3H]ouabain to neuronal Na +,K +-ATPase |
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