NZは交感神経ニューロンを解剖し、収穫の彼女を訓練するための研究室メンバーバースビスワス、アンドリュースプロウル、とライアンウィレットに感謝します。 NIH - NINDSからの補助金によって支え。

1。解剖のための準備： <ol><li>解剖する前に、実験の性質、およびラットテールコラーゲンでコーティングして24時間に応じて適切な培養皿を（10センチまたは6ウェルまたは24ウェルプレート、など）を選択します。ラットテールコラーゲンを調製し、コート培養皿にそれを使用するための方法は、（1）他の場所に記載されている。 </li><li>リン酸塩で0.25％トリプシン溶液を調製生理食塩水（PBS）（無EDTA）とフィルター滅菌はバッファ。 -20℃で1mlのアリコートとストアを準備する</li><li>蒸留/脱イオンH 2 O、2.5 mMのウリジンのそれぞれと5 - FDUを含む溶液を準備するろ過滅菌し、50μlのアリコートを準備。 -20℃で保存</li><li> 10％の熱不活化ウマ血清（熱が30分、56 º Cの水浴中でインキュベートすることによって不活性化）及び5％ウシ胎児血清を含むRPMI 1640培地：完全培地を準備します。 </li><li> 1％の熱不活化ウマ血清+ NGF（100ηg/ml最終濃度）+ペニシリン/ストレプトマイシン（1 / 250）+ 250分の1 uridine/5-FDU溶液（10μMの最終濃度）を含むRPMI 1640培地：最終的なメディアを準備します。 。注：すべての時間を使用する前に、この培地を新鮮で構成する必要があります。 24ウェルプレートフォーマットでの実験では、一つはこの培地12 mlのを準備しておく必要があります。それはあなたが少し余分に準備することをお勧めします。 NGFは、商業的供給源の多様性（このプロトコールの最後にある表を参照）から購入することができます。 </li><li>設備：一つは解剖顕微鏡と光源が必要になります。焦点のヒントとデュアルグースネック光ファイバー照明器が好ましい。ステンレス製のピンセットをマイクロは、解剖用の二つのペア、および解剖ハサミ（また、ステンレス鋼：加えて、一つは（オートクレーブまたは少なくとも20分間70％エタノールに浸漬することによって）、以下の解剖ツールを洗浄し、滅菌する必要があります。とRobozからの両方）。二つの滅菌注射針（26 gague × 1 ½インチまたは類似の）も必要になります。それは発泡スチロールの3&quot;× 3&quot;ピースアルミホイルに包んでキムワイプで覆われている解剖のボードを、設定する必要があります。除染する70％エタノールで基板にスプレーしてください。最後に、ファイアーポリッシュガラスの牧草地のピペットを準備。回転しながら火を研磨するためには、、ガラスピペットを取ると、数秒のための炎の先端を保持する。検査時に、先端は滑らかになりますし、開口部が狭くなります。これは、細胞が解離されるステップのために必要です。ピペットは、滅菌残るように細胞培養のフードで火災、研磨を行います。 </li></ol> 2。上頚神経節の解剖： <ol><li>年齢P0またはP1のものである新生児ラットの仔を収集する。 </li><li>急速に汚染の可能性を減らすために70％エタノールで解剖のために子犬を拭いてください。 </li><li>急速にハサミ（このステップは、ビデオには表示されません）と子犬斬る。簡潔に過剰な血液を排出するために滅菌ガーゼに対して滅菌ピンセットを用いて頭部を保持する。 </li><li>上に向けて腹側を解剖パッドとを志向尾方端にヘッドを置きます。切断された気管は容易に見えるはずです。パッドへの口の屋根も一つのピンを押すとパッドに断絶された気管かかわらずつ目のピンを押す：次の方法で解剖パッドに切断された頭部を保護するために無菌の注射針を使用してください。解剖顕微鏡の下にパッドを配置し、解剖を始める。 </li><li> 、気管の周りに両方鉗子（それぞれの手で1つ）離れて明確な皮膚や脂肪を使用して余りに深く行かないことに注意しながら。これを行うと、気管の両側に互いにほぼ平行に走って筋肉のペアを公開し​​ます。注：解剖時に、あなたが冷滅菌PBSまたは冷たい、過剰な血液を洗い流すために、エリアを清潔にし、潤いを保つために、滅菌、パスツールピペットで提供される無血清RPMI 1640培地で灌漑する必要があります。これは、に応じて、一度だけまたは複数回行うことができますどのように速くは動いて。経験の解剖のために、それは神経節のペアを分析する唯一の約2〜3分かかります。 </li><li>最初の側の筋肉を切断し、除去する鉗子を使用する。 </li><li>筋肉が削除されると、頚動脈が見えるようになります。多くの場合、それは光ピンク色であり、血液が含まれています。それはあなたの近くに表示されることがありますし、この理由のために頸動脈がすぐに顕著でないかもしれないこと、他の血管よりも色が薄く表示されることがあります。この動脈は、&quot;Y&quot;字型のリボンのようなものに気管および分岐すると一緒に動作します。この分岐は重要なランドマークであり、一度それが格納されている、それは、SCGを見つけることは比較的容易になります。 </li><li>ほとんどの尾方端に動脈を切断し、鉗子（もう一方の端がまだアタッチされている）とわずかに持ち上げます。一度これを行うには、THR緩くちょうど分岐の時点で、動脈に付着している組織のアーモンド形、光沢のある見て小さな質量を持って表示されます。プレおよび/または後の神経節の接続詞EADのような。これは、上頸神経節です。優しく冷たい、無血清RPMI 1640培地を含む小規模な培養皿（35ミリ）で、それと場所を分離するために他の手で鉗子の2番目のペアを使用してください。 </li><li>次に、同じプロトコルに続く気管の反対側に神経節を抽出する。 </li></ol> 3。ガングリオンのクリーニング： <ol><li>一度、すべての神経が出解剖されている、彼らは可能な限り余分な組織のを解放する必要があります。 </li><li>解剖顕微鏡の下で、神経は頸動脈、脂肪や他の組織片が接続されていることが表示される場合があります。これらの不要物を離れていじめるために、それぞれの手で鉗子を操作します。場所は、RPMI 1640培地を含む、滅菌15ミリリットルコニカル、ポリプロピレン管に神経節を掃除した。 </li><li>プレおよび/または後の神経節の接続詞はまた離れてトリミングする必要があります。各神経節は約10,000のニューロンを持っています。神経細胞が播種されると、彼らは彼らの神経突起を再生するだろう。 </li></ol> 4。交感神経ニューロンの文化の確立： <ol><li> 2分間200xgで神経を遠心し、上清を捨てる。 </li><li> 0.25％トリプシン溶液の0.5〜1 mlの神経を再懸濁します。 30分、37˚Cウォーターバスまたはインキュベーターに置きます。これは神経節に細胞を解離するのに役立ちます。 </li><li> 30分後、出希釈し、2分間200xgでトリプシンと遠心分離機を中和するために完全培地10 mlを加える。 </li><li>上清を捨て、最終的な培地2mlに再懸濁します。 </li><li>さらにファイアーポリッシュガラスの牧草地のピペットで上下に神経を摩砕して細胞を解離する。神経を20回ひいて粉にすると数分間チューブを氷上に置きます。 </li><li>一方、（直径約2 / 3 1 / 2〜mm）の開口部が狭いように、火研磨ピペットより。それが冷えるまで数分間待ってから、20倍以上の神経を砕いて粉にする。続行するように、培地は細胞が解離されていることを示す次第にcloudierになります。一度か二度以上の細胞が解離どの程度に応じてこの手順を繰り返します。いくつかの時間後、すべての神経節が神経細胞に解離があるとない無傷の神経はチューブで表示されていないことがわかります。解離しないいくつかの組織の材料があるかもしれません。この時点で、任意の非解離破片が底に沈降できるように、チューブを数分間氷中に放置。今慎重に別のチューブの上部と場所から、ほぼすべてのメディアのを収集する。あなたが解離していないゴミを収集しないことを保証するために、チューブの底に約50μlを残す。解離細胞を含むチューブに最終的な媒体の多くを追加：このボリュームは、培養プレートのいずれかの文化に神経細胞を使用しているのタイプによって異なります。たとえば、次のように、ウェル当たりの最終培地0.5 mlで、ウェルあたり24ウェルディッシュに採用される場合には、プレート0.5節（ウェルあたり〜10,000ニューロン）。典型的なネズミの糞には、24節を与えるだろう12匹、で構成されています。これは、種子1つの24ウェルプレートおよび最終的なメディアの総容量は12 mlのに十分です。注：Gangliaはまたグリアと文化に存在すると、他の細胞型の小集団が含まれています。最終的な培地でUridine/5-FDUを使用しても、その増殖を防止し、彼らの死を推進していきます。 </li><li>新鮮RPMI 1パーセントウマ血清+ NGFとuridine/5-FDUと培地の2 / 3を置き換えることにより、フィードセル毎に48時間。注：最初は、文化がない神経突起が付いている円形に見える交感神経ニューロンが含まれています。短い神経突起は、解剖後に表示されてから24時間となり、徐々に時間をかけて密度の高いとより多くの枝状になる。 </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

私たちは文化のためのSprague Dawleyラットを使用。 神経節を清掃時間と世話をする。すべての残骸、血管や脂肪を取り除きます。このステップでは、多かれ少なかれ均質と無関係な細胞型のない文化を確保するために重要です。 uridine/5-FDUの加算は、主として文化を汚染する、残りの（有糸分裂）非神経細胞を排除するか、少なくともそれらの増殖を抑制します。 さらに、解離工程の間、患者にな?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
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protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

ラットの上頚神経節（SCG）は交感神経ニューロンが含まれている小さな、光沢のある、アーモンド形の構造です。これらのニューロンは、頭頸部領域の交感神経神経支配を提供し、彼らは十分に特徴付けと、比較的均質な集団（4）を構成する。交感神経ニューロンは、生存、分化および軸索成長のための神経成長因子（NGF）に依存しており、NGFの広範な可用性は、自分たちの文化と実験的操作を容易に（2、3、6）。これらの理由から、培養交感神経ニューロンは、ニューロンの発達と分化、プログラムおよび病理学的細胞死のメカニズム、及びシグナル伝達（1、2、5、および6）を含む研究のさまざまな分野で使用されている。新生児ラットと培養交感神経ニューロンからSCGを解剖することは非常に複雑ではありませんし、かなり迅速に習得することができます。この記事では、新生児ラットの仔からSCGを分析すると、交感神経ニューロンの文化を確立するためにそれらを使用する方法を詳細に説明します。資料も準備手順と、これを達成するために必要な様々な試薬と装置を説明します。

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

これは、新生児ラットの仔の上頚神経節（SCG）からプライマリ交感神経ニューロンを分離して培養する方法を説明するプロトコルです。

ラット上頚神経節（SCG）から培養交感神経のためのプロトコル

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K ビュー.  Columbia University. これは、新生児ラットの仔の上頚神経節（SCG）からプライマリ交感神経ニューロンを分離して培養する方法を説明するプロトコルです。

ビデオ: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

マイクロ流体デバイスでコンパートメントのためのE18脳ラットのニューロンの準備

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

生物学

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

好中球単離プロトコル

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

コンパートメント文化の後根神経節ニューロンの準備とメンテナンス

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

ワームの研究室の紹介：培養ワームから変異へ

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

ワイルド毛虫から血液細胞を抽出するための単純なプロトコル

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

管細胞の収集培養初代ラットインナー髄

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

ラットの外側尾静脈から血液をサンプリング

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146の単離<sup&gt; +</supラット肺から&gt;常駐肺間葉系幹細胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

マウスからの膵島分離のための簡単な高効率プロトコル

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

機械評価のためのヒト筋学の上室から骨格筋生検の収集

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...