이 연구는 중국 국립 자연과학 재단(81673676)과 동관과학기술국(2019622101002)의 지원을 받았습니다. 저자는 메리 로즈 설리반 박사 (수술조교수, 하버드 의과 대학) 기술 컨설팅에 감사드립니다.

모든 실험 프로토콜과 동물 절차는 국가 연구 위원회의 윤리적 원칙 지침을 준수했다.
1. 재료 의 준비
<ol><li>실험을 수행하기 전에 자동 복제를 사용하여 모든 기기및 ddH2O를 멸균합니다. 기기에는 수술 용 가위, 안과 가위, 집게, 숟가락 핵 칸막이, 유리 접시 (직경 60-100mm) 및 유리 차단기를 포함하지만 이에 국한되지 않습니다.</li>
	<li>다음과 같이 Krebs 용액을 준비(표 1): 사용하기 전에 ddH2O와 함께 모든 화학 물질을 용해하십시오. CaCI2를 별도로 녹이고 침전물을 피하기 위해 교반하는 동안 혼합 용액에 천천히 추가합니다.</li>
	<li>10% 태아 소 혈청 (FBS) 및 1 % 항생제 / 항진균 (100x)F12 미디어로 구성된 린스 미디어를 준비하십시오. F12 미디어의 44.5 mL에 FBS 5mL 및 항생제/항마이코틱의 0.5 mL를 추가합니다.</li>
	<li>2% B-27, 1% FBS, 1% L-글루타민, 1% 항생제/항마이코틱(100x), 그리고 0.1% glia 유래 신경영양인(GDNF)을 포함하는 신경물질 A를 포함하는 신경 세포 A를 준비합니다. B-27의 200 μL, FBS 100 μL, L-글루타민 100 μL, 항생제/항진균제 100μL(100x), GDNF(10 μg/mL) 10μL를 신경분질 A 미디어의 9.5mL에 추가합니다. 참고: B-27, L-글루타민 및 GDNF의 신선도를 보장하기 위해 사용 후 1주일 이내에 뉴런 미디어 A를 준비합니다.</li>
	<li>뉴런 미디어 A의 준비와 동일한 절차를 사용 하 여 신경 미디어 B를 준비 하지만 FBS를 추가 하지 않고.</li>
	<li>소화용액 10 mg의 콜라게나아제 타입 II, 소 세럼 알부민 6mg, 산소 안정크렙스 용액 10mL에서 항생제/항진균(100x)의 200μL를 용해하여 소화용액 1을 준비한다.</li>
	<li>행크의 균형 잡힌 소금 용액 4mL로 0.25% 트립신 1mL을 희석하여 소화 용액 2를 준비합니다.</li>
	<li>코팅 된 커버립접시를 준비합니다.<ol>
<li>유리 커버립을 48웰 배양판에 넣을 때 라미나르 플로우 벤치에 집게를 사용하십시오.</li>
		<li>폴리-D-리신을 ddH2O로 희석하여 폴리-D-lysine 재고로 0.1 mg/mL농도로 희석합니다. 재고를 -20°C에 보관하고 사용하기 전에 해동합니다.</li>
		<li>각 커버슬립 위에 폴리-D-리신 스톡 40μL을 추가하고 실온에서 10분 동안 용액을 배양합니다. 참고: 기저 영역이 다른 플레이트의 경우 농도를 4 μg/cm2로조정합니다. 예를 들어, 24웰 플레이트로부터 1개의 우물의 기저면적이 2cm2인경우, 폴리-D-리신 재고의 80 μL을 잘 전달해야 한다. 각 cm2에는 폴리 D-리신 4μg가 포함됩니다.</li>
		<li>48웰 플레이트에서 폴리-D-리신을 제거하고, ddH2O 세 번으로 커버립을 헹구세요.</li>
		<li>플레이트가 무수성인지 확인하기 위해 최소 30분 동안 라미나르 플로우 후드로 플레이트를 건조시다.</li>
		<li>라미닌 코팅 전에 4°C에서 플레이트를 1일 동안 보관하십시오. 플레이트를 -20°C로 저장하는 것이 장기 보관에 바람직하다.</li>
		<li>4 °C에서 라미닌을 해동하십시오. 라미닌 육수로 50 μg/mL의 농도로 ddH2O로 라미닌을 희석시하십시오. 재고를 -20°C로 저장하고 사용하기 전에 4°C에서 해동하십시오.</li>
		<li>파이펫을 사용하여 희석된 라미닌 100μL을 각 커버슬립의 맨 위로 옮기고 라미닌을 4°C에서 1h로 배양한다. 참고: 기저 영역이 다른 플레이트의 경우 농도를 5 μg/cm2로조정합니다. 예를 들어, 24웰 플레이트로부터 하나의 우물의 기저 면적이 2cm2인경우, 희석된 라미닌200μL을 우물에 옮기는다. 각 cm2에는 라미닌 5μg가 포함됩니다.</li>
		<li>라미닌 용액을 제거하고 커버립을 ddH2O로 한 번 헹구는 다. 긁어방지를 위해 커버슬립 가장자리에서 이 작업을 수행합니다. 참고: 코팅된 커버립은 최대 2주 동안 4°C로 보관할 수 있습니다.</li>
	</ol>
</li>
</ol>2. 방광 수확
<ol><li>5주 된 스프라그-Dawley 쥐를 구하십시오.</li>
	<li>안정적인 산소 상태와 pH 수준에 도달하기 위해 얼음 욕조에서 적어도 30 분 동안 크렙스 용액에 카보겐 (95 % 산소, 5 % CO2)을주입합니다.</li>
	<li>자궁 경부 탈구를 통해 안락사 후, 살균을 위해 30 에탄올에 75 %의 쥐를 담그십시오.</li>
	<li>소독 수술 수건에 쥐를 놓고 복부를 노출. 복강을 열고 가위와 집게 세트로 방광을 드러냅니다.</li>
	<li>방광을 부드럽게 들어 올리고 방광목에서 방광을 다른 가위와 집게로 잘라 교차 오염을 방지합니다. 방광을 감기 산소 안정 크렙스 솔루션에 빠르게 배치하여 세포 생존을 향상시킵니다. 참고: 방광이 제거되면 다음 작업을 신속하게 수행하여 뉴런 생존 가능성을 개선하십시오.</li>
	<li>크렙스 용액을 세 개의 유리 요리와 유리 차단기에 추가합니다.</li>
	<li>프리쿨링을 위해 얼음 욕조에 크렙스 용액으로 유리 접시와 유리 차단기를 준비합니다.</li>
	<li>혼동을 방지하기 위해 해당 번호 1-3로 이러한 컨테이너를 표시합니다.</li>
	<li>각 유리 접시에 집게와 숟가락 핵 분배기와 함께 페어링합니다.</li>
	<li>유리 접시 1에서 안과 가위로 방광을 열고 집게와 숟가락 핵 칸막이로 펴놓습니다.</li>
	<li>방광을 유리 차단기 1에 헹신 다음 유리 접시 2에 놓습니다.</li>
	<li>유리 접시 2의 집게와 안과 가위를 사용하여 조직 표면에 부착 된 지방을 제거하십시오.</li>
	<li>방광을 유리 차단기 2에 헹신 다음 유리 접시 3에 놓습니다.</li>
	<li>집게와 숟가락 핵 분배기를 유리 접시 3에 사용하여 방광을 부드럽게 긁어 외인성 부착물을 제거합니다.</li>
	<li>방광을 유리 차단기 3에서 헹구고, 방광을 14mL의 콜드 크렙스 용액으로 15mL 원심분리기 튜브로 옮기고, 356 x g 및 4°C에서 1분 동안 샘플을 회전시합니다.</li>
	<li>오염을 줄이기 위해 크렙스 용액을 사용하여 두 개의 다른 튜브에서 이전 단계를 두 번 반복합니다.</li>
</ol>3. 2단계 방광 소화
<ol><li>원심분리기 관에서 1mL의 소화용액 1을 포함하는 2mL 바이알로 방광을 전달한다. 안과 가위를 사용하여 방광을 용액의 작은 조각 (1mm 보다 작은)으로 자른다.</li>
	<li>방광 용액을 멸균 세포 배양 접시(직경 100mm)에 9mL의 소화용액 1과 섞는다. 5% CO2,37°C 및 200 rpm 미만의 1시간 동안 흔들리는 인큐베이터에서 1단계 소화를 수행합니다.</li>
	<li>1단계 소화 후, 원심분리기는 용액을 4°C에서 356 x g에서 8분 동안 356 x g로 한다.</li>
	<li>예열을 위해 37°C 수조에 소화용액 2를 놓습니다.</li>
	<li>원심 분리 후, 소화 용액 1을 포함하는 상체를 제거하고 세포 퇴적물을 수확한다. 일부 남은 액체는 허용됩니다. 용액의 완전한 제거는 세포 손실을 촉진할 수 있다.</li>
	<li>15mL 원심분리기 튜브에 따뜻한 소화용액 2와 세포 침전물을 섞고 37°C 수조에서 소화하면서 혼합물을 흔들어 5분 동안 섞는다. 단계 2 소화의 7 분 이상하지 마십시오 또는 뉴런은 멸망한다.</li>
	<li>소화 후, 즉시 차가운 헹기 매체의 10 mL와 혼합물에 트립신을 비활성화합니다. 참고: 0°C-4°C에서 다음 단계를 수행합니다. 얼음 목욕은 그러한 상태를 제공 할 수 있습니다.</li>
	<li>원심분리 후 세포 퇴적물을 4°C에서 356 x g에서 8분 동안 수확하십시오. 남은 트립신이 세포 성장에 유해하기 때문에 가능한 한 많은 미디어를 제거하십시오.</li>
	<li>3mL의 뉴런 미디어로 퇴적물을 부드럽게 재중단합니다. 높은 생존율을 위해 세포를 포함하는 용액에서 기포가 생성되지 않았는지 확인합니다.</li>
	<li>70 μm 세포 스트레이너를 통해 혼합물 매체를 50mL 원심분리기 튜브로 필터링합니다.</li>
	<li>30 분 동안 얼음 목욕에 30 rpm에 셰이커에 여과물을 유지합니다. 이 단계는 필요하지 않지만 권장됩니다.</li>
	<li>4°C에서 356 x g에서 원심분리로 세포를 수거하고, 1mL의 뉴런 미디어 A에서 세포 펠릿을 부드럽게 재보냅니다.</li>
	<li>준비된 48웰 플레이트의 각 웰에 500 μL의 셀 혼합물을 넣습니다.</li>
	<li>37°C 및 5% CO2에서인큐베이터내의 배양 세포.</li>
	<li>모든 미디어를 1h의 뉴런 미디어 B로 교체하여 혈청없는 문화를 제공합니다.</li>
	<li>3일마다 뉴런 미디어 B의 절반을 변경합니다. 참고: 뉴런은 5-7일 간의 배양 후 면역 세포화학 실험을 위한 준비가 되어 있습니다.</li>
</ol>

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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>

저자는 큰 이해 상충을 선언하지 않습니다.

플레이트 준비 면역형광 또는 칼슘 이미징 실험을 위한 6-, 12 또는 48웰 배양 판에서 유리 커버립을 사용하는 것은 경제적이고 시료 절약 적인 동작입니다. 세포는 1 차적인 세포 배양의 준비 도중 커버립 없이 접시에서 잘 자랍니다. 따라서, 커버립은 서양 블롯 또는 폴리머라제 연쇄 반응과 같은 실험에서 분배할 수 있다. 또한, 코팅은 커버립의 유무에 관계없이 세포를 도금하기 전...

하부 요로에는 두 가지 주요 기능이 있습니다: 주기적인 소변 저장 및 배뇨1. 하부 요로 신경계(LUTNS)는 이러한 기능을 제어하고 많은 신경병증에 섬세하고 민감하며, 이는 선천적(포르피리아), 획득(라임병), 질병 상태(당뇨병 세포질), 약물 유도(출혈성 방광염), 수술 발생(복부 절제술 절제술, 4, 외상성 척추 상해, 4, 외상성 척추 상해, 4, 외상성 척추 손상, 4, 외상성 척추 손상, 3) 및 상해(외상성 척추 손상, 3) 및 상해(외상성 척추 손상, 5, 외상성 척추 손상,3) 생리적/병리학 연구에서 생체 내 및 체외 실험도 똑같이 중요합니다. LUTNS에 대한 생체 내 연구는 장기, 세포 및 분자 수준에서 한동안 수행되었지만, 오줌 방광에서 1 차 적인 뉴런에 대한 체외 연구는 거의 존재하지 않는8,9. 본 연구는 제한적이지만, 우리는 다른 연구자들이 그것을 개선할 수 있도록 이 지역에 있는 연구를 개척하기를 희망합니다. 이러한 방식으로, 이러한 공동 배양은 방광 뉴런 기능 장애와 같은 표현형에 있는 생리적 기능 장애의 세포 이해로 이끌어 낼 수 있습니다.
근육 세포의 명확한 방향성을 가진 장내 근육과 는 대조적으로, 방광의 근육은 조직되지 않습니다10. 따라서 방광의 외부 층을 벗겨내는 대신, 이 방법은 방광 전체를 소화하여 작동의 어려움을 줄이고 높은 세포 생존율에 대한 전처리 시간을 단축할 것을 제안합니다.
이 방법에 따라, 우리는 뉴런 과 다른 세포의 혼합 배양을 얻을 수 있습니다. 그들의 존재는 생체 내환경(11)을모방하기 때문에 다른 세포는 필수 불가결하다. 또한, 이러한 세포는 배지에서 사용할 수 없는 물질을 제공한다.
이 방법은 소화를 위한 두 단계를 포함합니다. 먼저, 콜라게나아제 형 II는 콜라겐을 가수분해하는 데 사용되며, 트립신이 이어서 조직을세포(10)로해리한다. 이런 식으로, 방광 조직은 단 하나 세포로 분산되고 그 때 상대적으로 독립적으로 성장합니다. 뉴런의 문화가 성숙할 때, 뉴런은 화상 진찰 또는 기능적인 소사에 사용될 수 있습니다.

<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

낮은 요로는 두 가지 주요 기능, 즉, 주기적인 소변 저장 및 배뇨; 이러한 기능은 중앙 및 말초 신경 조절을 통해 중재됩니다. 낮은 요로 신경계에 대한 광범위한 연구가 실시되었지만 대부분의 연구는 1 차 문화에 초점을 맞추고 있습니다. 이 프로토콜은 스프라그-Dawley 쥐에서 방광 뉴런과 glia의 격리 및 문화에 대한 방법을 소개합니다. 이 방법에서, 뉴런과 glia는 5-7 일 동안 37 °C, 5 % CO2 인큐베이터에서 배양되었다. 그 결과, 그(것)들은 관련 후속 면역 형광 실험에 적합한 성숙한 모양으로 성장했습니다. 세포는 광학 현미경을 사용하여 형태학적으로 관찰되었다. 뉴런, 시냅스 소포 및 glia는 각각 β-III-tubulin 및 MAP-2, 시냅신-1 및 GFAP 염색에 의해 확인되었다. 한편, 면역세포화학은 콜린 아세틸트랜스퍼라제, DYNLL2 및 SLC17A9와 같은 여러 신경전달물질 관련 단백질에서 수행되었다.

1 차 세포 배양 과정에서, 획득 된 세포는 부착 된 상태 전에 밝고 명확한 경계로 둥글게되었다. 뉴런이 성장함에 따라, 수상자와 축축은 구별되기 시작했다. 문화의 5-7 일 후에, 신경은 화상 진찰 또는 기능 연구 결과에 이상적이었던 긴 돌출을 가진 성숙한 양식에 도달했습니다. 대부분의 불순물 및 세포 파편은 변화하는 매체로 인해 제거될 수 있었지만, 폴리-D-리신 및 라미닌 코팅에 부착된 특?...

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

이 프로토콜은 추가 세포 실험을 위한 쥐 방광에서 1 차적인 신경세포 및 glia 격리를 위한 반복가능한 프로토콜을 설치하기 위하여 시도합니다.

성인 쥐 요로 방광에서 1 차신경및 glia의 격리 그리고 문화

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

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JoVE Journal

Neuroscience

3.8K 조회수.  Guangzhou University of Chinese Medicine. 이 프로토콜은 추가 세포 실험을 위한 쥐 방광에서 1 차적인 신경세포 및 glia 격리를 위한 반복가능한 프로토콜을 설치하기 위하여 시도합니다.

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

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

연구

신경과학

Bilaminar Co-culture of Primary Rat Cortical Neurons and 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 co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

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

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

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

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

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

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

<em&gt; 체외</em성인 마우스 Myenteric 신경총에서 분리 된 장내 뉴런과 교세포의&gt; 준비

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

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.

마우스 도파민 뉴런의 차 문화

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

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

병아리 배아에서 분리 및 해리 감각 뉴런의 문화

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

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

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

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

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

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 postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

신생아 생쥐의 척수 신경 세포 분리 및 배양

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 ...

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 ...