This project was funded by the Norwegian University of Life Sciences and the Research Council of Norway, grant number 243811 and 244461 (Aquaculture program). We are grateful to Lourdes Carreon Tan at the Norwegian University of Life Sciences for maintaining the fish facility.

The animal experiments performed in this study were approved by the Norwegian University of Life Sciences, following guidelines for the care and welfare of research animals.
1. Preparation of Solutions
<ol>
	<li>Calibrate the osmometer and pH meter instruments according to the manufacturer&#8217;s instructions to ensure correct measurements.</li>
	<li>Prepare 500 mL of Ca2+- and Mg2+-free Dulbecco&#8217;s phosphate-buffered saline (dPBS), adjusting the pH to 7.75 (step 1.2.1) and the osmolality to 290 mOsm/kg (step 1.2.2) before a sterile filtration (step 1.2.3).
	<ol>
		<li>Adjust the pH to 7.75 with a calibrated pH meter by carefully adding drops of a 1 M NaOH solution while carefully stirring the solution.</li>
		<li>Measure the osmolality of the solution using a calibrated osmometer and calculate the amount of mannitol needed to increase the osmolality to 290 mOsm/kg. Add the required amount of mannitol and then measure the osmolality of the solution again to ensure the correct adjustment. 
		NOTE: The amount of mannitol needed depends on the osmolality measured, which usually varies between different batches. One molecule of mannitol equals 1 osmotic particle.</li>
		<li>Sterile-filter the dPBS solution using a 0.2 &#181;m filter. 
		NOTE: The solution may be stored at 4 &#176;C for at least 3 months.</li>
	</ol>
	</li>
	<li>Prepare 500 mL of Leibovitz (L-15) culture medium without L-glutamine and bicarbonate, and supplement it with 10 mM NaHCO3 (step 1.3.1), 4.5 mM glucose (step 1.3.2), and 2 mM commercially available glutamine (step 1.3.3). Adjust the solution to 290 mOsm/kg (step 1.3.4), sterile-filter it (step 1.3.5), and add 2.5 mL of a penicillin-streptomycin solution (step 1.3.6). 
	NOTE: The solution may be stored at 4 &#176;C for at least 4 weeks.
	<ol>
		<li>Add 420 mg of NaHCO3 per 500 mL of culture medium to achieve a 10 mM solution.</li>
		<li>Add 405 mg of glucose per 500 mL of culture medium to achieve a 4.5 mM solution.</li>
		<li>Add 5 mL of 100x stock of a glutamine solution in 500 mL of culture medium to achieve a 2 mM solution. Stir until all solutes are dissolved.</li>
		<li>Adjust the solution to 290 mOsm/kg with mannitol using an osmometer (as performed in step 1.2.2).</li>
		<li>Perform a sterile filtration of the L-15 medium using a 0.2 &#181;m filter.</li>
		<li>After the sterile filtration, add 2.5 mL of a penicillin-streptomycin solution, corresponding to 50 U/mL of penicillin and 50 &#181;g/mL of streptomycin.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of a trypsin solution using 1 mg/mL of (0.1%) trypsin type II-S dissolved in the modified dPBS (prepared in step 1.2). Make aliquots of 2 mL in sterile plastic tubes and store them at -20 &#176;C until use. 
	NOTE: Aliquots of 2 mL can be stored at -20 &#176;C for at least 3 months.</li>
	<li>Prepare 50 mL of a trypsin inhibitor solution using 1 mg/mL of (0.1%) trypsin inhibitor type I-S, and supplement it with 2 &#181;g/mL of DNase I dissolved in the modified dPBS (prepared in step 1.2). Make aliquots of 2 mL in sterile plastic tubes and store them at -20 &#176;C until use. 
	Note: Aliquots of 2 mL can be stored at -20 &#176;C for at least 3 months.</li>
</ol>
2. Preparation of Equipment
<ol>
	<li>Prepare glass pipettes by fire&#160;polishing the tip for the cell dissociation. Make tips with 2 different size categories (a medium opening of 0.6&#8211;0.8 &#181;m and a small opening of 0.4&#8211;0.6 &#181;m) by playing with the time and the distance from the flame while simultaneously turning the pipette. Place the glass pipettes in a clean glass container and autoclave them (using the solid mode at 105 &#176;C for 45 min).</li>
	<li>Prepare a polystyrene box with ice.</li>
	<li>Prepare a plastic tube with 1.5 mL of modified dPBS (prepared in step 1.2) to transfer the pituitaries to during the dissection and keep it on ice.</li>
	<li>Prepare a 15 mL tube with 10 mL of modified dPBS (prepared in step 1.2) and keep it on ice until the cell dissociation.</li>
	<li>Prepare fresh or thaw aliquots of the trypsin and trypsin inhibitor solutions (prepared in steps 1.4 and 1.5) and keep them on ice until use.</li>
	<li>Set the water bath to 26 &#176;C before starting the dissection to allow the water to reach the desired temperature before the chemical dissociation of the pituitaries.</li>
	<li>Clean all dissection tools with 70% ethanol before, during, and after use, including fine forceps to use for the dissection and fine needles and a wax plate to pin the fish. Use clean gloves (change and clean frequently) during the entire procedure to avoid a potential contamination of the cells.</li>
</ol>
3. Pituitary Dissection and Cell Dissociation
<ol>
	<li>Euthanize the fish by immersing it in ice slush (0 &#176;C). Leave the fish in the ice slush for at least 1 min to ensure an irreversible hypothermic shock. 
	NOTE: Immobilization and equilibrium loss will occur after a few seconds. Make sure the fish is completely submerged in the ice water and does not lie on top of the ice, as the latter will lead to suffocation and potential skin burns instead of a rapid and humane hypothermic shock.</li>
	<li>Quickly transfer the fish in a net to a wax plate under the dissection microscope and destroy the spine by a needle punch through the neck.</li>
	<li>Pin the head of the fish to the wax plate using fine needles, by inserting one needle in the front (over the mouth) and one on each side of the head (behind the brain) to stabilize the head before the dissection.</li>
	<li>View the fish under the dissection microscope and gently scrape off the scales at the top of the head using fine forceps with an angled tip.</li>
	<li>Carefully insert the angled tip of the fine forceps under the skin from the side of the head and remove the skull roof by slowly moving the forceps in the direction of the mouth, while holding a firm grip to the forceps enclosing the skull roof.</li>
	<li>Cut the spinal cord completely off using forceps with an angled tip. 
	NOTE: Time is an important factor, so work efficiently and accurately to limit the time spent from the dissection of the pituitaries until the cells are plated in the dish. After some practice, pituitary dissection should take around 2&#8211;3 min per fish, of which only around 10 s are needed to pick the pituitary when the brain is already exposed.</li>
	<li>Carefully flip the brain over towards the mouth to expose the pituitary and dissect it with clean forceps with a straight tip. Transfer the pituitary to a plastic tube containing 1.5 mL modified dPBS (for details, see step 2.3) placed on ice.&#160;Check the tip of the forceps under the microscope to ensure that the pituitary is successfully added to the tube and nothing is left on the forceps. 
	NOTE: Dissect as many pituitaries as needed, depending on the number of culture dishes that are going to be prepared simultaneously. As a rule of thumb, calculate at least 10 pituitaries from adult fish per dish to have an appropriate density of cells (which depends on the downstream application).</li>
	<li>Spin down the pituitaries in a tabletop centrifuge for 1&#8211;2 s at room temperature. Carefully remove the dPBS using a glass pipette and take care to avoid removing any pituitaries.</li>
	<li>Add 1 mL of the trypsin solution (prepared in step 1.4) to wash the pituitaries, spin them down in a tabletop centrifuge for 1&#8211;2 s, and remove most of the liquid using a glass pipette before adding an additional 1 mL of trypsin solution.</li>
	<li>Incubate the tube in a water bath at 26 &#176;C for 30 min, preferably with gentle shaking, or alternatively, flick the tube a couple of times during the incubation period.</li>
	<li>Spin down the pituitaries in a tabletop centrifuge for 1&#8211;2 s at room temperature and make sure all pituitaries are located at the bottom of the tube. Remove most of the trypsin solution with a glass pipette and add 1 mL of the trypsin inhibitor solution (prepared in step 1.5) to inactivate the trypsin and wash the pituitaries.</li>
	<li>Collect the pituitaries at the bottom of the tube by spinning them down in a tabletop centrifuge for 1&#8211;2 s at room temperature. Remove most of the liquid with a glass pipette and add 1 mL of the trypsin inhibitor solution (prepared in step 1.5). Place the tube on a water bath at 26 &#176;C for 20 min, preferably with gentle shaking, or alternatively, flick the tube a couple of times during the incubation period to inactivate the trypsin.</li>
	<li>Prepare a 35 mm poly-d-lysine-coated cell culture dish with a central glass bottom by adding 2 mL of L-15 medium and letting that incubate for at least 10 min at 26 &#176;C and with 1% CO2 so that it can reach the right temperature and pH before plating the cells. 
	NOTE: A plastic cell culture dish can also be used. The main advantage of using a dish with a centralized glass bottom is that the area where the cells are plated is smaller, and therefore, fewer pituitary cells are required per dish. Some downstream applications might also require a glass bottom (i.e., some imaging techniques).</li>
	<li>Set the cooling centrifuge for the 15 mL tubes to 4 &#176;C to allow it to reach the desired temperature before starting the mechanical dissociation.</li>
	<li>Collect the pituitaries at the bottom of the tube by spinning down the tube with the trypsin inhibitor solution (from step 3.11) in a tabletop centrifuge for 1&#8211;2 s at room temperature and make sure all pituitaries are located at the bottom of the tube before carefully removing most of the trypsin inhibitor solution using a glass pipette. 
	Note: Perform all the following steps of the protocol in a laminar flow bench (LAF) certified for cell work to avoid a contamination of the cells.</li>
	<li>Add 1 mL of ice-cold modified dPBS (prepared in step 2.4) to the pituitary tissue pieces and gently suck the tissue pieces up and down (6&#8211;7x) in a fire-polished glass pipette (prepared in step 2.1). 
	NOTE: Temperature is an essential aspect, so keep all solutions at 4 &#176;C. From this point onward, perform all steps on ice when possible.</li>
	<li>Spin down the tissue pieces in a tabletop centrifuge for 1&#8211;2 s at room temperature and carefully transfer the upper part of the dPBS containing the dissociated cells (almost 1 mL) to a new 15 mL tube placed on ice. Avoid moving the pipette towards the bottom of the tube where the tissue pieces are left.</li>
	<li>Repeat step 3.16&#8211;3.17, dissociating the cells in 1 mL of ice-cold dPBS at a time, until all 10 mL of dPBS is used. Start using a glass pipette with a medium-sized opening and switch to a glass pipette with a smaller opening towards the end (for the last 3&#8211;4 mL of dPBS added) of the dissociation. If there are still visible tissue pieces towards the end of the dissociation procedure, increase the pipetting to approximately 10x towards the 2 last steps of dPBS for dissociation, and finally leave the residual tissue pieces behind. Resuspend the cells gently and avoid creating bubbles as it may cause cell damage when the cells attach to the surface of the bubbles. 
	NOTE: This is a critical step of this protocol and requires some training to achieve a good result.</li>
	<li>Balance the tube in the centrifuge (pre-cooled to 4 &#176;C) and spin down the cells at 200 x g for 10 min at 4 &#176;C. Make sure to mark the side of the tube where the cell pellet will be.</li>
	<li>Carefully remove the tube from the centrifuge and gently transfer the supernatant with a glass pipette to an empty 15 mL plastic tube directly after the centrifugation. Be sure to remove most of the supernatant but take care to avoid losing the small (often invisible) cell pellet.</li>
	<li>Carefully resuspend the cell pellet in 50&#8211;100 &#181;L of L-15 culture medium. 
	NOTE: At this step, it is possible to count the cells and/or perform a viability test (such as a cell counting in the presence of trypan blue using a hemocytometer), but beware that using part of the cell suspension for this purpose will lower the yield. Avoid using a large suspension volume as it will increase the risk of cells diffusing outside of the center of the dish.</li>
	<li>Carefully drip the dissociated cells in the middle of the cell culture dish containing 2 mL of the L-15 medium (prepared in step 3.13). Allow the cells to sink to the bottom of the dish for approximately 5 min before moving them to the incubator, to avoid having the cells spread outside of the sunken part of the glass bottom.</li>
	<li>Incubate the dish at 26 &#176;C and 1% CO2 to allow all the cells to sink thoroughly and attach to the bottom of the dish.</li>
	<li>After 30 min, look at the cells in the microscope to make sure they have attached to the culture dish.</li>
	<li>Continue to incubate the cells at 26 &#176;C and 1% CO2.</li>
	<li>Carefully change most of (but not all) the medium every 3&#8211;4 days. 
	Note: Avoid changing the medium more frequently, as it may disturb the cells and make them detach from the glass bottom. Use the cells for experiments within a week after seeding them.</li>
</ol>

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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. 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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. 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The authors have nothing to declare.

In vitro cell culture systems provide powerful tools for researchers to answer a plethora of different biological questions if used in the right way1. It is important to remember that dissociated cells that have lost their connections to the neighboring cells may have obtained different functional properties than they originally had in vivo. To avoid being at the risk of misinterpreting the results obtained from in vitro experiments, it is important to consider adjusting...

Cell culture is one of the main tools used in molecular biological research, providing an excellent model system for answering different biological questions ranging from normal cellular physiology to drug screening and carcinogenesis1. Primary cells, isolated directly from the animal tissue using enzymatic and/or mechanical methods,are often considered more biologically relevant than cell lines as the biological response may be closer to the in vivo situation. Protocols for preparing primary cell cultures should be optimized for each species and cell type of interest in order to mimic the characteristics to which a cell is adapted and obtain physiologically meaningful results.
Numerous protocols describe culture conditions for mammalian cell systems, while similar protocols describing primary culture conditions for fish cells are rather scarce in comparison. Cells are vulnerable to rapid changes in temperature, pH, and osmolality, and are particularly fragile during the dissociation procedure. Commercial salt solutions and culture media used for mammalian cell cultures are not optimal for teleost fish, especially in terms of pH buffer system(s) and osmolality. It is, therefore, important to measure and adjust the solutions to physiologically relevant levels of these parameters in the species of interest.
Primary pituitary cultures have been made from several teleost fish species, including common carp (Cyprinus carpio)2,3, grass carp (Ctenopharyngodon idella)4, goldfish (Carassius auratus)5, rainbow trout (Oncorhynchus mykiss)6, European eel (Anguilla anguilla)7, tilapia (Oreochromis mossambicus)8, zebrafish (Danio rerio)9, and Atlantic cod (Gadus morhua)10. Apart from adjusting the incubation temperature to the species of interest, several of these protocols have incubated the cells at mammalian-like conditions that may be suboptimal for the species of interest, with a pH from 7.2 to 7.5 in a humidified atmosphere containing 3 - 5% CO2. In addition, it is unclear if the osmolality of solutions used for preparing several of these primary cell cultures were adjusted and stable between different solutions.
The current protocol is based on previous work with primary cultures from Atlantic cod10 and comprises adjustments of incubation temperature, osmolality, pH, and pH buffer systems, including the partial pressure of carbon dioxide (pCO2), to the physiology of medaka (O. latipes). Medaka is a small (3&#8211;4 cm) freshwater fish, native to East Asia. These days, it is used as a model species in many research laboratories around the world, as it is relatively easy to breed and highly resistant to many common fish diseases11. There are several advantages of using medaka as a model, including a temperature tolerance from 4&#8211;40 &#176;C11, a short generation time, transparent embryos, a sex-determining gene12, and a sequenced genome13, as well as many other available genetic resources.
The primary culture conditions in this protocol are optimized to match the temperature of 26 &#176;C that medakas are kept at in the fish facility. Further, the osmolality is reduced from 320 mOsm/kg from Atlantic cod living in salt water to 290 mOsm/kg for medaka living in fresh water and is in accordance with the normal osmolality of medaka plasma14. In comparison, the typical osmolality of mammalian plasma is in the range of 275&#8211;295 mOsm15. Fish lives in a variety of temperatures and have gills that are in direct contact with water, making the pH and buffer capacity of the blood and extracellular fluid in fish different from those in mammals. Mammalian culture media usually include buffer systems that result in a pH of around 7.4 when the media are equilibrated to a standard atmosphere of 5% CO2 in humidified air at 37 &#176;C. The pH is temperature dependent and the value for neutral pH (in water) increases with a decreasing temperature16. Typical teleost fish plasma pH ranges from 7.7 to 7.917. The optimization of this protocol included a reduction from pH 7.85 for cod kept at 12 &#176;C to pH 7.75 for medaka kept at 26 &#176;C by increasing the CO2 from 0.5% to 1%.
In addition, the bicarbonate buffer capacity is quite different in fish and mammals. CO2 is easily exchanged over the gills in fish and the pCO2 in water is only a small fraction of the pCO2 in the lung18. Changing either the temperature or the pCO2 will change the pH and buffer of the medium. Consequently, neither the pH nor the pCO2 recommended for incubating mammalian cells is optimal for fish cells, and therefore, the culture media should be optimized with buffer systems containing physiologically relevant values for fish and the particular species of interest. This protocol describes how to prepare primary cell cultures from medaka pituitaries and include adjustments of the incubation temperature, osmolality, pH, and pH buffer system, in addition to other important parameters to consider when preparing primary cell cultures from non-mammalian species.

<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

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

This protocol describes the preparation of a primary cell culture from medaka pituitaries and provides healthy cells that can be maintained in a culture for at least one week. The protocol is based on physiological relevant values for medaka14 and is additionally optimized to the pituitary tissue in adult fish, using a pH of 7.75 and an osmolality of 290 mOsm/kg during the entire procedure from the tissue harvesting to the plated cells in culture (<strong class="xf...

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

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

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

preparation-high-quality-primary-cell-culture-from-fish

Research

JoVE Journal

Neuroscience

15.1K Views.  Norwegian University of Life Sciences. Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

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

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine 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 mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

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

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

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

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

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

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

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

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

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

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

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

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 accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

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

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

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

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

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

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...