Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Deze studie werd ondersteund door de National Natural Science Foundation of China (Grant no. 81673676) en Dongguan Science and Technology Bureau (Grant no. 2019622101002). De auteurs danken Dr. Maryrose Sullivan (Assistant Professor in Surgery, Harvard Medical School) voor technisch advies.

Alle experimentele protocollen en dierprocedures voldeden aan de ethische principes van de Nationale Onderzoeksraad.
1. Bereiding van materialen
<ol><li>Steriliseer alle instrumenten en ddH2O met behulp van een autoclaaf voordat u het experiment uitvoert. Instrumenten omvatten maar zijn niet beperkt tot chirurgische scharen, oogheelkundige scharen, tangen, lepels nucleus divider, glazen schalen (60-100 mm in diameter) en glasbrekers.</li>
	<li>Bereid de Krebs-oplossing als volgt v...

<ol>
	<li>Fowler, C. J., Griffiths, D., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+micturition.">The neural control of micturition.</a> Nature Reviews Neuroscience. 9 (6), 453-466 (2008).</li><li>Golbidi, S., Laher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+diabetes+mellitus.">Bladder dysfunction in diabetes mellitus.</a> Frontiers in Pharmacology. 1, 136 (2010).</li><li>Iwasawa, E., 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=Long-term+Effects+of+Intravenous+Cyclophosphamide+in+Combination+with+Mesna+Provided+Intravenously+and+via+Bladder+Perfusion+in+a+Patient+with+Severe+Multifocal+Motor+Neuropathy.">Long-term Effects of Intravenous Cyclophosphamide in Combination with Mesna Provided Intravenously and via Bladder Perfusion in a Patient with Severe Multifocal Motor Neuropathy.</a> Internal Medicine. 56 (14), 1893-1896 (2017).</li><li>Lange, M. M., van de Velde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+and+sexual+dysfunction+after+rectal+cancer+treatment.">Urinary and sexual dysfunction after rectal cancer treatment.</a> Nature Reviews. Urology. 8 (1), 51-57 (2011).</li><li>Lin, C. S., 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=Nerve+function+and+dysfunction+in+acute+intermittent+porphyria.">Nerve function and dysfunction in acute intermittent porphyria.</a> Brain. 131, 2510-2519 (2008).</li><li>Rantell, A., 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=What+is+the+utility+of+urodynamics,+including+ambulatory,+and+24+h+monitoring,+in+predicting+upper+urinary+tract+damage+in+neuro-urological+patients+and+other+lower+urinary+tract+dysfunction?+ICI-RS+2017.">What is the utility of urodynamics, including ambulatory, and 24 h monitoring, in predicting upper urinary tract damage in neuro-urological patients and other lower urinary tract dysfunction? ICI-RS 2017.</a> Neurourology and Urodynamics. 37, 25-31 (2018).</li><li>Halperin, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Lyme+neuroborreliosis.">Diagnosis and management of Lyme neuroborreliosis.</a> Expert Review of Anti-Infective Therapy. 16 (1), 5-11 (2018).</li><li>Walter, 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=Reliability+of+supraspinal+correlates+to+lower+urinary+tract+stimulation+in+healthy+participants+-+A+fMRI+study.">Reliability of supraspinal correlates to lower urinary tract stimulation in healthy participants - A fMRI study.</a> Neuroimage. 191, 481-492 (2019).</li><li>Leitner, L., 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=A+novel+infusion-drainage+device+to+assess+lower+urinary+tract+function+in+neuro-imaging.">A novel infusion-drainage device to assess lower urinary tract function in neuro-imaging.</a> BJU International. 119 (2), 305-316 (2017).</li><li>Smith, T. H., Ngwainmbi, J., Grider, J. R., Dewey, W. L., Akbarali, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in-vitro+preparation+of+isolated+enteric+neurons+and+glia+from+the+myenteric+plexus+of+the+adult+mouse.">An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse.</a> Journal of Visualized Experiments. (78), e50688 (2013).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Roppongi, R. T., Champagne-Jorgensen, K. P., Siddiqui, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Density+Primary+Hippocampal+Neuron+Culture.">Low-Density Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (122), e55000 (2017).</li><li>Arms, L., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptides+in+lower+urinary+tract+function.">Neuropeptides in lower urinary tract function.</a> Handbook of Experimental Pharmacology. (202), 395-423 (2011).</li><li>Moriyama, Y., Hiasa, M., Sakamoto, S., Omote, H., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+nucleotide+transporter+(VNUT):+appearance+of+an+actress+on+the+stage+of+purinergic+signaling.">Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.</a> Purinergic Signal. 13 (3), 387-404 (2017).</li><li>Chaudhury, A., He, X. D., Goyal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+plays+a+key+role+in+nitrergic+neurotransmission+by+transporting+nNOS&#945;+to+enteric+varicosity+membrane.">Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOS&#945; to enteric varicosity membrane.</a> American journal of physiology. Gastrointestinal and Liver Physiology. 301 (3), 498-507 (2011).</li><li>Le Berre-Scoul, C., 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=A+novel+enteric+neuron-glia+coculture+system+reveals+the+role+of+glia+in+neuronal+development.">A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.</a> The Journal of Physiology. 595 (2), 583-598 (2017).</li><li>Hayashi, H., Yamada, M., Kumai, J., Takagi, N., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+laminin-111-derived+peptide-chitosan+matrices+in+a+primary+culture+of+rat+cortical+neurons.">Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.</a> Archives of Biochemistry and Biophysics. 648, 53-59 (2018).</li><li>Bottenstein, J. E., Sato, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+rat+neuroblastoma+cell+line+in+serum-free+supplemented+medium.">Growth of a rat neuroblastoma cell line in serum-free supplemented medium.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (1), 514-517 (1979).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Survival+of+Hippocampal+Neurons+in+B27-Supplemented+Neurobasalm,+a+New+Serum-free+Medium+Combination.">Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasalm, a New Serum-free Medium Combination.</a> Journal of Neuroscience Research. 35 (5), 567-576 (1993).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1 (5), 2406-2415 (2006).</li><li>Georgas, K. 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=An+illustrated+anatomical+ontology+of+the+developing+mouse+lower+urogenital+tract.">An illustrated anatomical ontology of the developing mouse lower urogenital tract.</a> Development. 142 (10), 1893-1908 (2015).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single-cell+RNA-seq+artifacts:+a+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li></ol>

De Voorbereiding van de plaat Het gebruik van glasafdekkingslips in 6-, 12- of 48-putkweekplaten voor immunofluorescente of calciumbeeldvormingsexperimenten is een economische en monstersparende operatie. Cellen groeien goed in platen zonder afdeklips tijdens de bereiding van primaire celculturen. Daarom zijn coverslips overbodig in experimenten, zoals westerse vlek of polymerasekettingreactie. Bovendien is coaten een noodzakelijke stap voor het plateren van cellen, met of zonder afdeklips. Laminine e...

De onderste urinewegen hebben twee hoofdfuncties: periodieke urineopslag en mictie1. Het lagere urinewegen zenuwstelsel (LUTNS) controleert deze functies en is delicaat en vatbaar voor vele neuropathieën, die aangeboren (porfyrie), verworven (ziekte van Lyme), secundair aan ziektetoestanden (diabetische cystopathie), geneesmiddel geïnduceerd (hemorragische cystitis), chirurgie veroorzaakt (abdominoperineale resectie), of letsel veroorzaakt (traumatische dwarslaesie)2,3,4,5,6,7. In fysiologische/pathologische studies zijn in vivo en in vitro experimenten even belangrijk. Hoewel in vivo onderzoek naar LUTNS al enige tijd op orgaan-, cellulair en moleculair niveau wordt uitgevoerd, is in vitro onderzoek naar primaire neuronen uit de urineblaas bijna onbestaande8,9. Hoewel de huidige studie beperkt is, hopen we het onderzoek op dit gebied te pionieren, zodat andere onderzoekers het kunnen verbeteren. Op deze manier kan deze co-cultuur leiden tot een cellulair begrip van fysiologische disfunctie in fenotypes, zoals blaasneurondisfunctie.
In tegenstelling tot enterische spieren met een duidelijke directionaliteit van de spiercellen in discrete lagen, zijn de spieren van de blaas ongeorganiseerd10. Daarom stelt deze methode voor om in plaats van de buitenste laag van de blaas af te pellen, de hele blaas te verteren om de moeilijkheidsgraad van de werking te verminderen en de voorverwerkingstijd te verkorten voor een hoge celoverlevingssnelheid.
Volgens deze methode kunnen we een gemengde cultuur van neuronen en andere cellen verkrijgen. De andere cellen zijn onmisbaar omdat hun aanwezigheid een in vivo omgeving nabootst11. Bovendien leveren dergelijke cellen de stoffen die niet beschikbaar zijn in het medium.
Deze methode omvat twee stappen voor de spijsvertering. Ten eerste wordt collagenase type II gebruikt om collageen te hydrolyseren, gevolgd door trypsine, om het weefsel te dissocieren in cellen10. Op deze manier worden blaasweefsels verspreid in enkele cellen en groeien ze vervolgens relatief onafhankelijk. Wanneer de cultuur van neuronen rijpt, kunnen de neuronen worden gebruikt voor beeldvorming of functionele assays.

<table border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-III-tubulin)</td>
			<td>Affinity</td>
			<td>AF7011</td>
			<td>ICC</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

De onderste urinewegen hebben twee hoofdfuncties, namelijk periodieke urineopslag en mictie; deze functies worden gemedieerd door middel van centrale en perifere neuroregulatie. Hoewel uitgebreid onderzoek naar het zenuwstelsel van de onderste urinewegen is uitgevoerd, hebben de meeste studies zich gericht op primaire cultuur. Dit protocol introduceert een methode voor de isolatie en cultuur van blaasneuronen en glia van Sprague-Dawley ratten. Bij deze methode werden de neuronen en glia gedurende 5-7 dagen geïncubeerd in een couveuse van 37 °C, 5% CO2. Als gevolg hiervan groeiden ze uit tot volwassen vormen die geschikt waren voor gerelateerde daaropvolgende immunofluorescentie-experimenten. Cellen werden morfologisch waargenomen met behulp van een optische microscoop. Neuronen, synaptische blaasjes en glia werden geïdentificeerd door respectievelijk β-III-tubuline en MAP-2, Synapsin-1 en GFAP-kleuring. Ondertussen, immunocytochemie werd uitgevoerd op verschillende neurotransmitter-gerelateerde eiwitten, zoals choline acetyltransferase, DYNLL2, en SLC17A9.

Tijdens het proces van primaire celkweek waren de verworven cellen rond met heldere en duidelijke grenzen vóór de bijgevoegde toestand. Naarmate de neuronen groeiden, begonnen dendrieten en axonen zich te onderscheiden. Na 5-7 dagen cultuur bereikten de neuronen een volwassen vorm met lange projecties, die ideaal waren voor beeldvorming of functiestudies. Hoewel de meeste onzuiverheden en celresten konden worden verwijderd als gevolg van veranderende media, waren bepaalde resten van poly-D-lysine en laginecoating zicht...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Dit protocol probeert een herhaalbaar protocol vast te stellen voor primaire neuronen en glia-isolatie van rattenblaas voor verdere cellulaire experimenten.

Isolatie en cultuur van primaire neuronen en Glia van volwassen rat urineblaas

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection en Primaire Cultuur van de embryonale Mouse hippocampus en de corticale neuronen

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Bilaminar Co-cultuur van primaire Rat corticale neuronen en Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Isolatie en Cultuur van hippocampale neuronen van Prenatal Muizen

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

Een<em&gt; In-vitro</em&gt; Voorbereiding van Geïsoleerde enterische neuronen en Glia uit de myenterische Plexus van de Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Primary Culture of Mouse Dopaminergic Neurons

Primaire Cultuur van de Muis dopaminerge neuronen

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

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Isolatie en Cultuur van Gedissocieerde sensorische neuronen van Chick Embryo

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

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

Isolatie, Cultuur en onderhoud op lange termijn van de Primary mesencefale dopaminerge neuronen uit embryonale knaagdieren Brains

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

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Isolatie en cultuur van de volwassen neurale stamcellen van de muis Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Spinal Cord Neurons Isolatie en Cultuur van Neonatale Muizen

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Generatie van neuro sferen van gemengde primaire Hippocampal en corticale neuronen geïsoleerd van E14-e16 Sprague Dawley rat embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...