Name: preparation of a high-quality primary cell culture from fish pituitaries
Uploaded: 2018-08-28T14:45:00
Description: Watch this Scientific Journal Video about Preparation of a High-quality Primary Cell Culture from Fish Pituitaries at JoVE.com

Dette projekt blev finansieret af norsk University of Life Sciences og Norge forskning-Rådet, tilskud antal 243811 og 244461 (akvakultur program). Vi er taknemmelige til Lourdes Carreon Tan på norsk University of Life Sciences for at opretholde fisk facilitet.

Dyreforsøg udført i denne undersøgelse blev godkendt af norske University of Life Sciences, følgende retningslinjer for pleje og dyrevelfærd, forskning.
1. forberedelse af løsninger
<ol><li>Kalibrere osmometer og pH meter instrumenterne ifølge producentens anvisninger til at sikre korrekte målinger.</li>
	<li>
Forberede 500 mL af Ca2 +- og Mg2 +-gratis Dulbeccos fosfatbufferet saltopløsning (dPBS), justering af pH til 7,75 (trin 1.2.1) og osmolalitet ti...

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. , (2010).</li><li>Ribeiro, L. P., Ahne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cell+culture:+initiation+of+a+line+of+pituitary+cells+from+carp+(Cyprinus+carpio)+to+study+the+release+of+gonadotropin+in+vitro.">Fish cell culture: initiation of a line of pituitary cells from carp (Cyprinus carpio) to study the release of gonadotropin in vitro.</a> In Vitro. 18 (5), 419-420 (1982).</li><li>Ribeiro, L., Ahne, W., Lichtenberg, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+normal+pituitary+cells+of+carp+(Cyprinus+carpio)+for+the+study+of+gonadotropin+release.">Primary culture of normal pituitary cells of carp (Cyprinus carpio) for the study of gonadotropin release.</a> In Vitro. 19 (1), 41-45 (1983).</li><li>Wong, A. O., Ng, S., Lee, E. K., Leung, R. C., Ho, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatostatin+inhibits+(d-Arg6,+Pro9-NEt)+salmon+gonadotropin-releasing+hormone-+and+dopamine+D1-stimulated+growth+hormone+release+from+perifused+pituitary+cells+of+chinese+grass+carp,+Ctenopharyngodon+idellus.">Somatostatin inhibits (d-Arg6, Pro9-NEt) salmon gonadotropin-releasing hormone- and dopamine D1-stimulated growth hormone release from perifused pituitary cells of chinese grass carp, Ctenopharyngodon idellus.</a> General and Comparative Endocrinology. 110 (1), 29-45 (1998).</li><li>Chang, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+pituitary+cell+dispersion+method+and+primary+culture+system+for+the+studies+of+gonadotropin-releasing+hormone+action+in+the+goldfish,+Carassius+auratus.+I.+Initial+morphological,+static,+and+cell+column+perifusion+studies.">Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. I. Initial morphological, static, and cell column perifusion studies.</a> General and Comparative Endocrinology. 77 (2), 256-273 (1990).</li><li>Weil, C., Hansen, P., Hyam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+pituitary+cells+in+primary+culture+to+study+the+secretion+in+rainbow-trout+-+Setting+up+and+validating+the+system+as+assessed+by+its+responsiveness+to+mammalian+and+salmon+gonadotropin-releasing+hormone.">Use of pituitary cells in primary culture to study the secretion in rainbow-trout - Setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin-releasing hormone.</a> General and Comparative Endocrinology. 62 (2), 202-209 (1986).</li><li>Montero, M., LeBelle, N., Vidal, B., Dufour, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+dispersed+pituitary+cells+from+estradiol-pretreated+female+silver+eels+(Anguilla+anguilla+L):+Immunocytochemical+characterization+of+gonadotropic+cells+and+stimulation+of+gonadotropin+release.">Primary cultures of dispersed pituitary cells from estradiol-pretreated female silver eels (Anguilla anguilla L): Immunocytochemical characterization of gonadotropic cells and stimulation of gonadotropin release.</a> General and Comparative Endocrinology. 104 (1), 103-115 (1996).</li><li>Xu, S. H., Cooke, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+of+tilapia+prolactin+cells.">Voltage-gated currents of tilapia prolactin cells.</a> General and Comparative Endocrinology. 150 (2), 219-232 (2007).</li><li>Lin, S. W., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+gonadotropins+(FSH+and+LH)+and+growth+hormone+(GH)+by+neuroendocrine,+endocrine,+and+paracrine+factors+in+the+zebrafish-An+in+vitro+approach.">Differential regulation of gonadotropins (FSH and LH) and growth hormone (GH) by neuroendocrine, endocrine, and paracrine factors in the zebrafish-An in vitro approach.</a> General and Comparative Endocrinology. 160 (2), 183-193 (2009).</li><li>Hodne, K., von Krogh, K., Weltzien, F. A., Sand, O., Haug, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+conditions+for+primary+culture+of+pituitary+cells+from+the+Atlantic+cod+(Gadus+morhua).+The+importance+of+osmolality,+pCO2,+and+pH.">Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO2, and pH.</a> General and Comparative Endocrinology. 178 (2), 206-215 (2012).</li><li>Wittbrodt, J., Shima, A., Schartl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medaka+-+a+model+organism+from+the+far+East.">Medaka - a model organism from the far East.</a> Nature Reviews Genetics. 3, 53-64 (2002).</li><li>Matsuda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMY+is+a+Y-specific+DM-domain+gene+required+for+male+development+in+the+medaka+fish.">DMY is a Y-specific DM-domain gene required for male development in the medaka fish.</a> Nature. 417 (6888), 559-563 (2002).</li><li>Kasahara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medaka+draft+genome+and+insights+into+vertebrate+genome+evolution.">The medaka draft genome and insights into vertebrate genome evolution.</a> Nature. 447 (7145), 714-719 (2007).</li><li>Miyanishi, H., Inokuchi, M., Nobata, S., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past+seawater+experience+enhances+seawater+adaptability+in+medaka,+Oryzias+latipes.">Past seawater experience enhances seawater adaptability in medaka, Oryzias latipes.</a> Zoological Letters. 2 (12), (2016).</li><li>Waymouth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolality+of+mammalian+blood+and+of+media+for+culture+of+mammalian+cells.">Osmolality of mammalian blood and of media for culture of mammalian cells.</a> In Vitro. 6 (2), 109-127 (1970).</li><li>Cameron, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+homeostasis+-+past+and+present+perspectives.">Acid-base homeostasis - past and present perspectives.</a> Physiological Zoology. 62 (4), 845-865 (1989).</li><li>Claiborne, J. B., Edwards, S. L., Morrison-Shetlar, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+regulation+in+fishes:+cellular+and+molecular+mechanisms.">Acid-base regulation in fishes: cellular and molecular mechanisms.</a> Journal of Experimental Zoology. 293 (3), 302-319 (2002).</li><li>Perry, S. F., Tufts, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish respiration. , (1998).</li><li>Hildahl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+tracing+of+luteinizing+hormone+&#946;-subunit+gene+expression+using+green+fluorescent+protein+transgenic+medaka+(Oryzias+latipes)+reveals+a+putative+novel+developmental+function.">Developmental tracing of luteinizing hormone &#946;-subunit gene expression using green fluorescent protein transgenic medaka (Oryzias latipes) reveals a putative novel developmental function.</a> Developmental Dynamics. 241 (11), 1665-1677 (2012).</li><li>Strandab&#248;, R. A. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+involved+in+GnRH2-stimulation+of+identified+LH-producing+gonadotropes+from+lhb-GFP+transgenic+medaka+(Oryzias+latipes).">Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes).</a> Molecular and Cellular Endocrinology. 372 (1-2), 128-139 (2013).</li><li>Verbalis, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+volume+regulation+in+response+to+changes+in+osmolality.">Brain volume regulation in response to changes in osmolality.</a> Neuroscience. 168 (4), 862-870 (2010).</li></ol>

In vitro celle kultur systemer giver kraftfulde værktøjer for forskere at besvare et væld af forskellige biologiske spørgsmål, hvis de anvendes i den rigtige vej1. Det er vigtigt at huske at dissocierede celler, der har mistet deres forbindelser til de nærliggende celler kan have opnået forskellige funktionelle egenskaber end de oprindeligt havde in vivo. For at undgå at blive for at fejlfortolke resultater fra in vitro- forsøg, er det vigtigt at overveje at jus...

Cellekultur er en af de vigtigste værktøjer, der anvendes i molekylær biologisk forskning, giver en fremragende modelsystem for at besvare forskellige biologiske spørgsmål spænder fra normale cellulær fysiologi stof screening og carcinogenese1. Primærelementer, isoleret direkte fra animalsk væv ved hjælp af enzymatiske og/eller mekaniske metoder er ofte betragtes som mere biologisk relevante end cellelinjer som den biologiske respons kan være tættere på i vivo situation. Protokoller for at forberede primære cellekulturer skal optimeres for hvert art og celle type af interesse for at efterligne de egenskaber, som en celle er tilpasset og opnå fysiologisk meningsfulde resultater.
Mange protokoller beskrive dyrkningsbetingelserne for pattedyr celle systemer, mens lignende protokoller der beskriver primære dyrkningsbetingelserne for fisk celler er temmelig sjælden i sammenligning. Celler er sårbare over for hurtige ændringer i temperatur, pH og osmolalitet og er særligt skrøbelige under proceduren dissociation. Kommercielle saltopløsninger og medier anvendt til pattedyr cellekulturer er ikke optimalt for teleost fisk, især hvad angår pH buffer system(er) og osmolalitet. Det er derfor vigtigt at måle og justere løsninger til fysiologisk relevante niveauer af disse parametre med arterne af interesse.
Primære hypofyse kulturer har gjort fra flere teleost fiskearter, herunder almindelig karpe (Cyprinus carpio)2,3, græs karper (Ctenopharyngodon idella)4, guldfisk (Carassius auratus )5, Regnbueørred (Oncorhynchus mykiss)6, europæisk ål (Anguilla anguilla)7, tilapia (Oreochromis mossambicus)8, zebrafisk (Danio rerio)9, og Torsk (Gadus morhua)10. Bortset fra justering inkubation temperatur for arter af interesse, har flere af disse protokoller inkuberes celler i pattedyr-lignende forhold, der kan være suboptimal for arter af interesse, med en pH-værdi fra 7,2 til 7,5 i en fugtig atmosfære der indeholder 3-5% CO2. Derudover er det uklart, om osmolalitet af løsninger, der anvendes til at forberede flere af disse primære cellekulturer blev justeret og stabil mellem forskellige løsninger.
Den nuværende protokol er baseret på tidligere arbejde med primære kulturer fra torsk10 og omfatter justeringer af inkubation temperatur, osmolalitet, pH buffersystemer, herunder partialtrykket af kuldioxid (pCO2), og pH til fysiologi af medaka (O. latipes). Medaka er en lille (3-4 cm) ferskvandsfisk, oprindelig i Østasien. Disse dage, bruges det som en model art i mange forskningslaboratorier verden over, da det er relativt let at opdrætte og meget modstandsdygtig over for mange almindelige fisk sygdomme11. Der er flere fordele ved at bruge medaka som en model, herunder en temperaturtolerance fra 4-40 ° C11, en kort generationstid, gennemsigtig embryoner, en sex-bestemmende gen12, og en sekventeret genom13, samt mange andre tilgængelige genetiske ressourcer.
De primære kultur betingelser i denne protokol er optimeret til at matche temperatur på 26 ° C, at medakas holdes på i fisk facilitet. Yderligere, osmolalitet reduceres fra 320 mOsm/kg fra torsk lever i saltvand til 290 mOsm/kg for medaka lever i ferskvand og er i overensstemmelse med den normale osmolalitet medaka plasma14. Til sammenligning er den typiske osmolalitet af pattedyr plasma i rækken af 275-295 mOsm15. Fiske liv i en bred vifte af temperaturer og har gæller, som er i direkte kontakt med vand, hvilket gør pH og buffer kapaciteten af blod og ekstracellulære væske i fisk forskellige fra dem i pattedyr. Pattedyr kultur medier omfatter normalt buffersystemer, der resulterer i en pH-værdi omkring 7,4, når medierne er ekvilibreres til en standard atmosfære med 5% CO2 i befugtet luft ved 37 ° C. PH er temperatur afhængige og værdien for neutral pH (i vand) stiger med en faldende temperatur16. Typiske teleost fisk plasma pH spænder fra 7,7 til 7,917. Optimering af denne protokol omfattede en reduktion fra pH 7,85 for torsk holdt på 12 ° C til pH 7,75 for medaka holdt ved 26 ° C ved at øge CO2 fra 0,5% til 1%.
Derudover er bikarbonat buffer kapaciteten helt anderledes i fisk og pattedyr. CO2 er let udveksles over gæller i fisk og pCO2 i vand er kun en lille brøkdel af pCO2 i lunge18. Ændre enten temperatur eller pCO2 ændrer pH og buffer af mediet. Derfor hverken pH eller pCO2 anbefales til inkubere pattedyrceller er optimal for fisk celler, og derfor kultur medier bør optimeres med buffersystemer indeholdende fysiologisk relevante værdier for fisk og den særlige arter af interesse. Denne protokol beskriver hvordan du forbereder primære cellekulturer fra medaka pituitaries og omfatter justeringer af inkubation temperatur, osmolalitet, pH og pH buffersystem, ud over andre vigtige parametre til at overveje, når du forbereder primære celle kulturer fra ikke-pattedyr arter.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (dPBS), without calcium chloride and magnesium chloride</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>D8537</td>
			<td>Adjust solution to pH 7.75 with 1M NaOH and 290 mOsm with mannitol.</td>
		</tr>
		<tr>
			<td>L-15 medium (Leibovitz), witout L-glutamine</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>L5520</td>
			<td>Supplement 500 ml culture medium with 10mM NaHCO3, 4.5 mM glucose, 2 mM Glutamax. Adjust solution to 290 mOsm with mannitol and filter solution through a 0.2 &#181;m PES sterile filter, before adding 2.5 ml Penicillin-Streptomycin solution (see below for details).</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>S5881</td>
			<td>Add drops of 1M solution to increase pH of dPBS and culture medium to 7.75.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>S5761</td>
			<td>10 mM NaHCO3 equals 420 mg per 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>63565</td>
			<td>Use to increase osmolality of dPBS and culture medium. Calculate correct amount needed to reach an osmolality of 290 mOsm.</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>G5400</td>
			<td>4.5 mM&#160; D-glucose equals 405 mg per 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco (Life Technologies, Paisley, UK)</td>
			<td>35050-061</td>
			<td>Alternative to L-glutamine, with increased stability. 2 mM Glutamax equals 5 ml of 100X stock in 500 ml culture medium.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>P0781</td>
			<td>Stock solution 10,000 units penicillin and 10 mg streptomycin per mL. Use 2.5 ml of stock solution in 500 ml L-15 medium (equivalent of 50 U/ml Penicillin and 50 &#181;g/ml Streptomycin).</td>
		</tr>
		<tr>
			<td>Trypsin type II-S</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>T7409</td>
			<td>Prepare 1 mg/ml in dPBS solution.</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor type I-S</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>T6522</td>
			<td>Prepare 1 mg/ml in dPBS solution, supplement with 2 &#181;g/ml Dnase I (see details below).</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma (Merck, Damstadt, Germany)</td>
			<td>D5025</td>
			<td>Use in trypsin inhibitor solution (see above).</td>
		</tr>
		<tr>
			<td>0.2 &#181;m Polyethersulfone (PES) sterile filter system</td>
			<td>Corning Inc. (Corning, NY)</td>
			<td>431097</td>
			<td>Use for sterile filtration of dPBS and L-15 medium after adjustments.</td>
		</tr>
		<tr>
			<td>35 mm cell culture dish with glass bottom, poly d-lysine coated</td>
			<td>MatTek Corporation (Ashland, MA)</td>
			<td>P35GC-1.5-10-C</td>
			<td>Can also be replaced by plastic dish, depending on downstream application.</td>
		</tr>
		<tr>
			<td>Dumont #5 fine forceps</td>
			<td>Fine Science Tools (CA)</td>
			<td>11254-20</td>
			<td>Straigt tip</td>
		</tr>
		<tr>
			<td>Dumont #5/45 fine forceps</td>
			<td>Fine Science Tools (CA)</td>
			<td>11253-25</td>
			<td>Angled 45&#176; tip</td>
		</tr>
		<tr>
			<td>Stereo microscope SZ61</td>
			<td>Olympus Corp. (Tokyo, Japan)</td>
			<td></td>
			<td>Use for dissection of pituitaries.</td>
		</tr>
		<tr>
			<td>Alegra X-22R Centrufuge</td>
			<td>Beckman Coulter Inc. (Brea, CA)</td>
			<td></td>
			<td>With cooling option (similar to current model XR-30).</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Techne (Staffordshire, UK)</td>
			<td></td>
			<td>Any water bath with the possibility of adjusting temperature will do.</td>
		</tr>
		<tr>
			<td>Pasteur glass pipettes</td>
			<td>VWR (NY)</td>
			<td>612-1701</td>
			<td>Outer diameter 1.6 mm, fire polish and autoclave before use.</td>
		</tr>
		<tr>
			<td>Galaxy MiniStar table centrifuge</td>
			<td>VWR (NY)</td>
			<td>521-2844</td>
			<td>Any small table centrigue will do.</td>
		</tr>
		<tr>
			<td>Fine needles / insect pins</td>
			<td>Fine Science Tools (CA)</td>
			<td>26001-40</td>
			<td>Diameter 0.03 mm. Other fine needles can be used instead.</td>
		</tr>
		<tr>
			<td>Wax plate</td>
			<td></td>
			<td></td>
			<td>Custom made by adding melted paraffin wax in large petri dish.</td>
		</tr>
	</tbody>
</table>

preparation of a high-quality primary cell culture from fish pituitaries

Primære cellekultur er et kraftfuldt værktøj, der er almindeligt anvendt af forskere til at studere cellulær egenskaber og mekanismer af isolerede celler i et kontrolleret miljø. Trods store forskelle i fysiologi mellem pattedyr og fisk, er primære celle kultur protokoller fra fisk ofte baseret på pattedyr dyrkningsbetingelser, ofte med kun mindre ændringer. Miljømæssige forskellene berører ikke kun kropstemperatur, men også blodserum parametre såsom osmolalitet, pH buffer kapacitet og pH. Celle Kulturmedier og lignende arbejder løsninger er beregnet til at efterligne Karakteristik af den ekstracellulære væske og/eller blod serum som en celle er tilpasset, er det af afgørende betydning, at disse parametre er tilpasset specielt til det pågældende dyr.
Den nuværende protokol beskriver optimeret primære dyrkningsbetingelserne for medaka (Oryzias latipes). Protokollen indeholder detaljerede trin til at isolere og opretholde sunde dissocieres hypofyse celler for mere end en uge og omfatter følgende trin: 1. justering af osmolalitet værdier fundet i medaka blodplasma, 2. tilpasning af de inkubation temperaturen til normal medaka temperatur (her i akvarium anlægget), og 3. justering af pH og bikarbonat bufferen til værdier sammenlignes med andre fiskearter, der lever på lignende temperaturer. Resultaterne præsenteres ved hjælp af den beskrevne protokol fremme fysiologisk meningsfulde resultater for medaka og kan bruges som en referenceguide af forskere at gøre primære cellekulturer fra andre ikke-pattedyr arter.

Denne protokol beskriver forberedelse af en primær cellekultur fra medaka pituitaries og giver sunde celler, der kan opretholdes i en kultur for mindst en uge. Protokollen er baseret på fysiologiske relevante værdier for medaka14 og er desuden optimeret til hypofyse væv i voksne fisk, med en pH på 7,75 og en osmolalitet 290 mOsm/kg under hele proceduren fra væv høst til den forgyldt cellerne i kulturen (figur 1 og <strong class=...

Watch this Scientific Journal Video about Preparation of a High-quality Primary Cell Culture from Fish Pituitaries at JoVE.com

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Her beskriver vi en protokol for at forberede og vedligeholde primære hypofyse cellekulturer fra medaka (Oryzias latipes). De optimerede betingelser i denne protokol tage vigtige parametre såsom temperatur, osmolalitet og pH ved at efterligne de fysiologiske tilstande af fisk, hvorved fysiologisk mere meningsfulde resultater.

Forberedelse af en høj kvalitet primære cellekultur fra fisk Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Research

JoVE Journal

Neuroscience

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

Isolering og dyrkning af neurale kamceller fra embryonale Murin Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Primary Culture of Mouse Dopaminergic Neurons

Primær Kultur Mouse dopaminerge neuroner

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Erhvervelse af højkvalitets digital video af<em&gt; Drosophila</em&gt; Larvestadiet og voksne opførsel fra en Lateral Perspektiv

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Isolation, Kultur og opretholdelse på lang sigt af Primary mesencephaliske dopaminerge neuroner af embryonale Gnavere Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Udarbejdelse af Primære Mixed Glia kulturer fra Voksen Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Low-Density Primary Hippocampal Neuron Culture

Low-Density Primær Hippocampal neuronkultur

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Live Imaging af primære hjernebarken celler ved hjælp af en 2D kultur

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Simpel Generation af højtydende kultur af inducerede neuroner fra menneskelige voksen hud fibroblaster

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

En rustfri protokol for høj kvalitet RNA isolering fra Laser fange Microdissected Purkinje celler i den menneskelige Post Mortem lillehjernen

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

Primær cellekultur af renset GABAergic eller Glutamatergic neuroner etableret gennem fluorescens-aktiveret celle sortering

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...