本研究は中国国立自然科学財団(グラント81673676)と東莞科学技術局(グラント2019622101002)によって支援されました。著者らは、メアリーローズ・サリバン博士(ハーバード大学医学部外科助教授)に技術コンサルティングをしてくれたことに感謝している。

すべての実験プロトコルと動物の手順は、国家研究評議会の倫理原則ガイドラインに準拠しています。
1. 材料の準備
<ol><li>実験を行う前に、オートクレーブを使用してすべての機器とddH2Oを殺菌します。器械は外科用はさみ、眼科用はさみ、鉗子、スプーン核の分け器、ガラス皿(直径60-100 mm)、およびガラスブレーカに限定されない。</li>
	<li>以下のようにクレブス溶液を調製する(表1):使用前にddH2Oと一緒にすべての化学物質を溶解します。CaCI2 を別々に溶解し、沈み込みを避けるために攪拌しながら混合溶液にゆっくりと加えます。</li>
	<li>10%の牛血清(FBS)と1%の抗生物質/抗ミコティック(100x)でF12培地で構成されるリンス培地を準備します。F12培地44.5mLにFBS5 mLと抗生物質/抗菌薬0.5mLを加えます。</li>
	<li>2%B-27、1%FBS、1%L-グルタミン、1%抗生物質/抗ミキ酢(100x)、および0.1%グリア由来神経栄養因子(GDNF)を有する神経基底A培地を調製する。B-27の200 μL、FBSの100 μL、L-グルタミンの100 μL、抗生物質/抗抗抗ミキティックの100 μL(100x)、および9.5mLのGDNF(10 μg/mL)の10 μLを神経基底A培地に加える。 注:B-27、L-グルタミンおよびGDNFの鮮度を確保するために使用の1週間以内にニューロンメディアAを準備してください。</li>
	<li>ニューロンメディアAの調製と同じ手順を用いてニューロンメディアBを準備するが、FBSを加えることなく。</li>
	<li>20mgのコラゲナーゼII型、6mgの牛血清アルブミン、200μLの抗生物質/抗ミキサン(100x)を酸素安定クレブス溶液10mLに溶解して消化液1を調製した。</li>
	<li>0.25%トリプシンの1 mLをハンクのバランスの取れた塩溶液の4 mLで希釈して消化液2を調製する。</li>
	<li>コーティングされたカバーリップでプレートを準備します。<ol>
<li>ガラスカバーリップを48ウェルの培養プレートに入れる場合は、層流のベンチで鉗子を使用します。</li>
		<li>ポリD-リジンストックとして0.1 mg/mLの濃度にddH2OでポリD-リジンを希釈します。在庫を–20°Cで保管し、使用前に解凍してください。</li>
		<li>各カバースリップの上にポリD-リジンストック40 μLを加え、室温で10分間インキュベートします。 注:異なる基底領域を持つ別のプレートの場合は、4 μg/cm2に濃度を調整します。たとえば、24ウェルプレートからの1ウェルの基底面積が2cm2の場合、80μLのポリD-リジンストックをウェルに移す必要があります。各cm2 は4μgのポリD-リジンを含んでいる。</li>
		<li>48ウェルプレートでポリD-リジンを取り除き、ddH2O thriceでカバーリップをすすいでください。</li>
		<li>プレートが無水であることを確認するために、少なくとも30分間ラミナーフローフードでプレートを乾燥させます。</li>
		<li>ラミニンコーティングの前に4°Cでプレートを保管してください。プレートを−20°Cで保管することは、長期保存に適しています。</li>
		<li>ラミニンを4°Cで解凍する。 ラミニンを薄い50 μg/mLの濃度にddH2Oで希釈します。在庫を-20°Cで保管し、使用前に4°Cで解凍してください。</li>
		<li>ピペットを使用して、希釈したラミニンを各カバースリップの上部に100 μL、4°Cで1時間インキュベートラミニンを1時間移動します。 注:異なる基底領域を持つ別のプレートの場合は、5 μg/cm2に濃度を調整します。例えば、24ウェルプレートからの1ウェルの基底面積が2cm2の場合、希釈したラミニンを200μLのウェルに移します。各cm2 は5μgのラミニンを含んでいる。</li>
		<li>ラミニン溶液を取り出し、ddH2Oでカバーリップを一度すすい取ります。スクレイピングを避けるために、カバースリップの端にこの操作を実行します。 注:コーティングされたカバーリップは、せいぜい2週間4°Cで保管することができます。</li>
	</ol>
</li>
</ol>2. 膀胱の収穫
<ol><li>5週齢のスプレイグ-ドーリーラットを入手する。</li>
	<li>安定した酸素状態とpHレベルに到達するために、氷浴中の少なくとも30分間、クレブス溶液にカルボゲン(95%酸素、5%CO2)を注入します。</li>
	<li>頸椎脱臼による安楽死後、ラットを殺菌のために30sの75%エタノールに浸す。</li>
	<li>殺菌した外科用タオルの上にラットを置き、彼らの腹部を露出させる。腹腔を開き、はさみと鉗子のセットで膀胱を明らかにする。</li>
	<li>膀胱をそっと持ち上げ、膀胱頸部から膀胱を別のハサミと鉗子で切り取り、クロスコンタミネーションを避けます。細胞の生存率を向上させるために、冷たい酸素安定クレブス溶液に膀胱を迅速に置きます。 注:膀胱を取り除いたら、ニューロン生存の見通しを改善するために、次の操作を迅速に行います。</li>
	<li>3つのガラス皿とガラスブレーカにクレブス溶液を加えます。</li>
	<li>ガラス皿とガラスブレーカを氷浴でクレブス溶液で準備して、予冷します。</li>
	<li>これらのコンテナに 1 ~ 3 の番号を付けて、混乱を防ぎます。</li>
	<li>各ガラス皿を鉗子とスプーン核仕切りと組み合わせる。</li>
	<li>ガラス皿1では、眼科用はさみで膀胱を切り開き、鉗子とスプーン核仕切りで開きます。</li>
	<li>ガラスブレーカ1で膀胱をすすいで、ガラス皿2に入れる。</li>
	<li>ガラス皿2の鉗子および眼科用はさみを使用してティッシュ表面の付着脂肪を除去する。</li>
	<li>ガラスブレーカ2で膀胱をすすい、ガラス皿3に入れる。</li>
	<li>鉗子とスプーン核仕切り器をガラス皿3に使用して膀胱を静かにこすり、外因性の付着物を取り除く。</li>
	<li>ガラスブレーカ3で膀胱をすすい、14 mLの冷たいクレブス溶液を用いた15mL遠心管に膀胱を移し、356 x g および4°Cで1分間サンプルを回す。</li>
	<li>上記の手順を、他の 2 つのチューブで 2 回、他の 2 つのチューブで 2 回繰り返して、汚染を減らします。</li>
</ol>3. 二段階膀胱消化
<ol><li>1 mLの消化液1を含む2mLバイアルに遠心管から膀胱を移す。眼科用はさみを使用して、膀胱を溶液中の小片(1mm未満)に切ります。</li>
	<li>膀胱溶液を9 mLの消化液1と滅菌細胞培養皿(直径100mm)で混合する。5%CO2、37°C、および200rpm下で1時間の振盪インキュベーターでステップ1消化を行います。</li>
	<li>ステップ1の消化後、溶液を4°Cで356xgで8分間遠心分離する。</li>
	<li>消化液2を予熱用の37°C水浴に入れる。</li>
	<li>遠心分離後、消化液1を含む上清を除去し、細胞沈降物を収穫する。残りの液体の一部は許可されています。溶液の完全な除去は、細胞の損失を促進する可能性があります。</li>
	<li>細胞沈積物と温かい消化液2を15 mL遠心分離管に混ぜ、37°Cの水浴で5分間消化しながら混合物を振る。ステップ2消化またはニューロンの7分を超えないで滅びる。</li>
	<li>消化後、10 mLのコールドリンス培地を混合したトリプシンを直ちに非アクティブ化する。 メモ:0°C~4°Cで以下の手順を実行します。 氷浴はそのような状態を提供することができます。</li>
	<li>遠心分離後の細胞沈込を4°Cで4°Cで8分間収穫します。残りのトリプシンは細胞増殖に有害であるため、できるだけ多くの培地を取り除きます。</li>
	<li>3 mLのニューロン培地で沈澱を緩やかに中断する。高い生存率のために細胞を含む溶液中に気泡が生成されないようにしてください。</li>
	<li>70 μmのセルストレーナーを介して、50 mL遠心分離チューブに混合培地をフィルターします。</li>
	<li>30rpmのシェーカーで30分間氷浴に入れ、濾過液を保管します。この手順は必須ではありませんが、推奨されます。</li>
	<li>4°Cで356xgで8分間遠心分離して細胞を採取し、ニューロン培地Aの1mLで細胞ペレットを静かに再懸濁する。</li>
	<li>調製した48ウェルプレートの各ウェルに500μLの細胞混合物を加えます。</li>
	<li>培養細胞は、37°Cおよび5%CO2で培養器中に含まれる。</li>
	<li>すべての培地を1時間でニューロン培地Bに交換し、無血清培養を行う。</li>
	<li>ニューロン培地Bの半分を3日ごとに変更します。 注意:ニューロンは、培養5~7日後に免疫細胞化学実験を行う準備ができています。</li>
</ol>

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著者らは、大きな利益相反を宣言していない。

プレートの準備 免疫蛍光またはカルシウムイメージング実験用の6-、12、または48ウェル培養プレートでのガラスカバーリップの使用は、経済的でサンプル節約的な操作です。細胞は、一次細胞培養の調製中にカバースリップのないプレートでよく成長する。そのため、カバーリップは、ウェスタンブロットまたはポリメラーゼ連鎖反応などの実験で欠かせない。さらに、コー?...

下部尿路には、尿の定期的な保存と、1のミチュリンの2 つの機能があります。下部尿路神経系(LUTNS)はこれらの機能を制御し、繊細で多くの神経障害の影響を受けやすく、先天性(ポルフィリン症)、後発性疾患状態(糖尿病性膀胱障害)、薬物誘発(出血性膀胱炎)、または外傷(脊髄損傷)2,3,4,6,7, 7, 7, 7, 7, 7, 7,7, 7.生理学的/病理学的研究では、生体内およびインビトロ実験も同様に重要である。LUTNSに関するインビボ研究は、しばらくの間、臓器、細胞、および分子レベルで行われてきたが、尿膀胱からの一次ニューロンに関するインビトロ研究はほとんど存在しない8,9である。今回の研究は限られていますが、他の研究者が改善できるように、この分野の研究を開拓したいと考えています。このようにして、この共培養は、膀胱ニューロン機能不全などの表現型における生理学的機能不全の細胞的理解につながる可能性がある。
離散層への筋肉細胞の明確な方向性を持つ腸筋とは対照的に、膀胱の筋肉は組織化されていない10である。したがって、膀胱の外層を剥離する代わりに、この方法は、手術困難を低減し、高い細胞生存率のための前処理時間を短縮するために膀胱全体を消化することを提案する。
この方法に従って、ニューロンと他の細胞の混合培養を得ることができる。その存在は生体内環境11を模倣するので、他の細胞は不可欠である。また、このような細胞は、培地で利用できない物質を提供する。
この方法は、消化のための2つのステップを伴う。まず、コラゲザーゼII型は、コラーゲンを加水分解するために使用され、続いてトリプシンが、組織を細胞10に解化する。このようにして、膀胱組織は単一細胞に分散され、その後比較的独立して成長する。ニューロンの培養が成熟すると、ニューロンは、イメージングまたは機能的アッセイに使用することができます。

<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

下部尿路は、2つの主要な機能、すなわち、定期的な尿貯蔵およびミチュリションを有する。これらの機能は、中央および末梢神経調節を介して仲介されます。低尿路神経系に関する広範な研究が行われているが、ほとんどの研究は、原発性文化に焦点を当ててきた。このプロトコルは、スプレイグ-ドーリーラットからの膀胱ニューロンとグリアの分離と培養のための方法を導入します。この方法では、ニューロンおよびグリアを37°C、5%CO2インキュベーターで5〜7日間インキュベーターでインキュベートした。その結果、それらは関連する後続の免疫蛍光実験に適した成熟した形状に成長した。細胞は光学顕微鏡を用いて形態学的に観察した。ニューロン、シナプス小胞、およびグリアは、それぞれβ-III-管状およびMAP-2、シナプシン-1、およびGFAP染色によって同定された。一方、免疫細胞化学は、コリンアセチルトランスビセラーゼ、DYNLL2、SLC17A9などのいくつかの神経伝達物質関連タンパク質に対して行われました。

一次細胞培養の過程で、獲得した細胞は、付着状態の前に明るく明確な境界で丸くした。ニューロンが成長するにつれて、樹状突起と軸索は明確になり始めました。5~7日間培養した後、ニューロンは長い投影で成熟した形に達し、イメージングや機能研究に最適でした。不純物および細胞デブリの大部分は、メディアの変化により除去することができたが、ポリD-リジンおよびラミニンコー?...

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

このプロトコルは、一次ニューロンとラット膀胱からのグリア分離のための反復可能なプロトコルを確立し、さらなる細胞実験を試みる。

成人ラット尿膀胱からの一次ニューロンとグリアの分離と培養

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

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K ビュー.  Guangzhou University of Chinese Medicine. このプロトコルは、一次ニューロンとラット膀胱からのグリア分離のための反復可能なプロトコルを確立し、さらなる細胞実験を試みる。

ビデオ: 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とマウス胎児海馬および皮質ニューロンの初代培養

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

神経科学

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.

初代ラット皮質ニューロンとグリアのBilaminarの共培養

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.

胎児マウスから海馬ニューロンの単離および培養

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.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

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.

マウスドーパミン作動性ニューロンの初代培養

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.

ニワトリ胚から解離感覚ニューロンの単離および培養

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.

胚性齧歯類の脳から単離、プライマリ脳ドーパミンニューロンの文化と長期メンテナンス

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.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

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.

新生児マウスからの脊髄ニューロンの単離と培養

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.

胚性マウス小脳から単離されたニューロンの一次培養

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 ...