Funding for the production of this manuscript was provided by Nottingham Trent University. The authors wish to thank the University yard manager, Anna Gregory for the use of her horses and provision of fecal samples for use in the protocol. Thanks also to Chester Zoo Wildlife Endocrinology Laboratory for use of their facilities.

倫理文：フィールドサンプリングおよび動物対象を含む手順は、ノッティンガム・トレント大学の動物、農村環境科学（ARES）科で承認されています。 糞便サンプルの1コレクション注：糞便サンプルおよびメタノールを取り扱う際は手袋を着用する必要があります。動物が人獣共通感染症に苦しんでいることができることを強い疑いがある場合は、そのような白衣などの防護服も着用してください。 <ol><li>排便、次の（数時間に分以内）できるだけ早く糞便サンプルを収集し、サンプルバッグに入れます。約10グラムの総重量で任意の排便サンプルの3つから5つのサブサンプルを取ります。 、収集の時刻と日付で袋にラベルを付け、個々のID、および分析まで-20℃でサンプルを凍結します。 </li><li>エラーを最小限に抑えるために、全てのサンプルは-20℃で凍結します °Cまでの研究である完全な単一のバッチとして分析のために一緒にサンプルを収集します。&lt;/李&gt; </ol> 2.糞便ホルモンの抽出<ol><li>室温で解凍し糞便サンプルを残します。 </li><li>サンプルは手動で独自のサンプルバッグ内の各サンプルを混合することにより均質化されていることを確認。 </li><li>各サンプルについて、その後8ミリリットルのガラスバイアルに糞を転送計量ボートに天秤に糞便材料で（0.003グラム±）0.50重量を量ります。各試料についての新しい計量ボートを使用して、各試料間の30％メタノールでピンセットをクリーニングします。 </li><li>糞便物質の0.5グラム、90％メタノール5mlでコンバイン：水、そして室温でオービタルシェーカー上でO / Nを振ります。 </li><li>翌朝は、サンプルをボルテックスした後、600グラムで20分間遠心します。 </li><li> 37℃の温度に設定した水浴中のラックに16×125 mm 2のガラス管と、所定の位置にメタノール画分をデカント。ドラフトチャンバー内の空気を使用して乾燥するまで蒸発させます。抽出用容器をすすぎ、処分のための医療廃棄物袋に残った糞石を配置します。 </li><li> 、100％で1ミリリットルのメタノールを糞便抽出物を再懸濁し、12×75ミリメートル2ポリプロピレンチューブに移します。すぐに分析するまで-20℃でキャップや店舗。 </li></ol> 3.ホルモン分析<ol><li>ポリクローナルコルチコステロンCJM006抗血清を用いて、酵素結合イムノアッセイ（EIA）との分析を行います。 注：第二EIAは（コルチゾールR4866抗血清を試験したが、これは（4.1節を参照してください）​​ポリクローナルコルチコステロンCJM006抗血清およびコルチゾールR4866抗血清とそれぞれのHRPのがCJマンロー、カリフォルニア大学によって供給されている生化学的検証ステップを達成するために失敗しました、デイビス、カリフォルニア</li><li>繰り返しのピペットと50μlのピペットチップを使用して、コーティング緩衝液（0.05 M炭酸水素ナトリウム、pHは9.6）で15,000、50μl/ウェルで96ウェルマイクロタイタープレートをロード：1で抗血清を希釈します。プレートシーラーでプレートを覆い、4℃でO / Nインキュベートします。非特異的結合ウェル（Nを表現するためにコーティングされていない2つの井戸を残しますSB）。 </li><li>使用直前に自動マイクロタイタープレート洗浄機（; 0.15MのNaCl、0.05％ツイーン20洗浄液）を用いてマイクロタイタープレートを5回洗浄します。 </li><li>千PG / - NSBのためのウェルあたり50μlのと「ゼロ」のウェル[EIA緩衝液（0.1 MのNaPO 4、0.149 MのNaCl、0.1％ウシ血清アルブミン、pH7.0）でのみ]、標準（コルチコステロン、3.9でプレートをロードウェル）または糞便抽出試料（適切に1時20分EIAバッファーで希釈し、4.1節を参照してください）​​。標準とゼロはそれぞれ、三重および四重に実行する必要があります。希釈した糞便抽出試料とNSBのは、二重に実行する必要があります。 </li><li> 7万：50μlの/ウェルを加え1にEIA緩衝液で希釈したラベル西洋ワサビペルオキシダーゼコンジュゲートᵻですぐにこれに従ってください。 </li><li> RTで2時間、暗所でマイクロタイタープレートをインキュベートします。 </li><li>洗浄溶液でマイクロタイタープレートを5回洗浄し、100μl/ WELと暗闇の中で、室温でプレートをインキュベートRT基板のL [0.4mMの2,2&#39;-アジノ-ジ（3-エチルベンズチアゾリンスルホン酸）ジアンモニウム塩、1.6mMのH 2 O 2、0.05 Mクエン酸、pHを4.0]。使用直前に基板を準備します。 </li><li>マイクロタイタープレートを読み取るために405nmの波長で分光光度計を使用してください。ゼロウェルの光学濃度が0.8〜1.0に達したときに、プレートのインキュベーションが完了したと見なされます。 </li><li>再現性を保証するために、約30％（EIA規格）、50％（生物学的サンプル、 例えば、国内の馬の糞便抽出液のプール）、70％（EIA規格）に結合するためにEIA緩衝液で希釈したコントロール[EIA規格または生物学的サンプルを使用]は、それぞれ&lt;10％の変動と&lt;15％の内およびアッセイ間の係数を実証します。 </li></ol> 4.酵素結合免疫測定法：研究種の検証とセックス<ol><li>生化学的検証（並列処理） <ol><li> EIA緩衝液を使用して、プールされた糞便余分に連続希釈を行いますCT、理想的には、いくつかの個体から疑われる高および低ホルモン濃度のサンプルから採取した（範囲：ニート、1：8192：2〜1）および第3章で説明したようにEIA上で実行されます。 注：糞便抽出物の連続希釈が標準曲線と直線回帰の結果に変位曲線を平行に得たときに並列処理が達成されたと見なされはR2&gt; 0.90、スロープ&gt; 0.50およびp &lt;0.05を示しています。標準曲線に50％前後を結合する段階希釈糞便抽出物は、すべての糞便抽出物を実行するために理想的な希薄化を考慮すべきである（ 例えば、1時20分）。線形回帰の結果はR2&gt; 0.90およびp &lt;0.05を実証するときにアッ​​セイに考え干渉はありません。 </li></ol></li><li>生化学的検証（干渉） <ol><li>適当に希釈したプールされた糞便抽出液200μlで連続的に希釈した標準液200μl（ゼロ、3.9〜1000 ngの/ウェルコルチコステロン）のスパイク[50％に希釈したparalleliによって決定バインディングSM（1時20分）]およびEIA分析プロット次のセクション3のようにEIA上で実行観察濃度マイナス予想濃度対バックグラウンド（ゼロサンプルの濃度）。 注：標準の希釈プールされた糞便抽出物の添加が有意量予想と線形回帰の結果を変更しない場合はR 2&gt; 0.90、1.0およびp &lt;0.05に近い傾斜をもたらします。 EIAとの干渉が達成されていません。 </li></ol></li><li>生物学的検証： 注：選択されたEIAは、関心のホルモンの生物学的に関連する変化を測定する能力を持っていることを実証[ 例えば、薬理以下のグルココルチコイド濃度の上昇（ACTHは、多くの場合、ライセンス手続きである）または生物学的課題（ 例えば、心理学的および/ ​​または環境;日和見無秩序なイベントである可能性がより高いです）]。 <ol><li>グルココルチコイド代謝物濃度のために糞便サンプルを選択し、抽出して分析します挑戦的なイベントの前と後の3 - セクション1に詳述プロセスを使用して。 注：挑戦的なイベントは、特定の種​​になります。例示的な方法は、国内の馬のために以下に提供されます。馬のための挑戦的なイベントの完全な例は、管理9と飼育手順16の使用を含め、以前に発表されています。挑戦的なイベント次のグルココルチコイド代謝物濃度の有意な上昇が観察され、統計的分析を用いて確認する必要があります。これは、適切な場合には反復測定は、サンプルの日付と個々の効果を組み込む必要があります。生物学的検証は、次に達成と考えられています。 </li></ol></li></ol>

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The authors have nothing to disclose.

糞便のコルチコステロン分析は、ウマにおいて副腎活動の長期的パターンを評価する手段を提供します。方法の非侵襲的な性質は、唾液、血漿分析9を含む副腎活性を評価するために使用される他のサンプリング方法の交絡効果を克服します。自由変動の馬を研究する場合に加えて技術は明確な非侵襲的な利点を有しています。 このメソッドとその適切な利用に?...

この方法は、副腎活性の非侵襲的評価を提供するために馬の糞便中のコルチコステロン濃度を分析することを目的とします。視床下部 - 下垂体 - 副腎（HPA）軸の活性を測定すると、キャプティブと国内の両方の種において潜在的に嫌悪事態への対応を研究するための一般に認められた手法です。参考技術と最も広く使用される方法は、しかしながら、例えば、糞便分析などの別の方法は、遊離の範囲の種を監視する能力を採血自体によって誘発されるストレスを克服し、可能にするために開発されてきた血漿1の使用です。 嫌悪状況の間に、生理的恒常性が破壊されています。下垂体前葉に作用し、副腎皮質刺激ホルモン（ACTH）の放出を刺激ホルモン放出ホルモン（CRH）の副腎皮質脳のリリースでは視床下部。 ACTHは血流に入り、種を分泌する副腎皮質を刺激しますsの特定のグルココルチコイド（GC）。グルココルチコイドは密接ではなく、一貫したがって、彼らはしばしば他のストレスリンクのホルモン2の上に優先的に測定されているすべてのエネルギーの高まりの状態で製造されるよりも、ストレスの多いイベントにリンクされています。グルココルチコイドは、馬のいくつかの適応効果を担当しています。エネルギーが急速に酸素摂取量を増加させ、脂肪酸およびグルコースの形で体内の貯蔵部位から動員され、感覚機能は、3と、血流を向上さ動き4に必要でない領域まで低下します。同様に対処メカニズムとして機能するように、グルココルチコイドの応力誘起される上昇はまた、次のストレッサー5のために動物を準備するのを助けることができます。 血漿および唾液中のホルモンのレベルを評価することは、糞便中の代謝物の測定は、ホルモンの代謝最終産物を測定し、しかし実際の循環ホルモンを測定することを含みます。循環ステロイドはリットルに異化されています彼らは腸のトラック6内細菌叢の酵素活性によって容易にさらなる変化を受ける胆汁に排泄前にアイバー。したがって、血液グルココルチコイドに向けイムノアッセイは、糞便、グルココルチコイド代謝物7の分析に適していないかもしれません。 糞便収集が馬に乱れを行うことができるように、コルチコステロンのための糞の分析は、多くの状況でHPA活性をモニターするために広く使用されています。馬の糞便中の上昇コルチコステロンは、術後獣医の治療8の間にあり、限定ハウジング9に含む潜在的嫌悪状況に応じて報告されています。糞便サンプルは、時間をかけてプールされたグルココルチコイドレベルではなく、血漿および唾液長期的に監視することが適切で作る、慢性または季節的パターン10によって提供される時間サンプリング点を反映しています。非侵襲性に起因し方法の性質は、サンプルを捕捉または拘束具11を必要とせずに、個々の繰り返し収集することができます。サンプリングプロトコルを計画する際しかし、種特異的腸通過時間を考慮しなければなりません。馬では、腸の通過時間は約18時間12したがって、副腎応答およびその後のコルチコステロン代謝物は1日HPA軸の初期起動後に糞便中に検出することができます。 非侵襲的なイムノアッセイ技術を利用する場合検討さ種について注意深く検証は13不可欠です。また、ホルモンの代謝物の排泄の性差が原因で、マウス14、鶏15を含む様々な種に排泄コルチコステロン代謝物の代謝速度とタイプの違いにおそらく報告されています。アッセイは目に詳述されているように男性と女性の国内の馬の両方で使用するために検証されたそのため、この方法の一部として重要でした電子プロトコル。男女間のホルモン代謝におけるこの違いは、アドレス指定されず、アッセイの検証の一部として含まれているまれまだデータ品質に対する影響を有しています。 この非侵襲的方法は、国内のウマにおける副腎活動の長期評価を可能にします。プロトコルの詳細分析の検証およびアッセイ技術自体の両方。

<table>
	<tbody>
		<tr>
			<td>Corticosterone antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Cortisol antibody &#38; HRP kit</td>
			<td>Coralie Munro - UC Davis</td>
			<td>NA</td>
			<td>No longer available through UC Davis - please see Arbor Assays</td>
		</tr>
		<tr>
			<td>Corticosterone synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>50-23-7</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Cortisol synthetic standard hormone</td>
			<td>Sigma Aldrich</td>
			<td>15087-01-1</td>
			<td>Harnful if ingested or with skin contact. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>67-56-1</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>144-55-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>497-19-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>7558-79-4</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma Aldrich</td>
			<td>10049-21-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>9048-46-8</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>9005-64-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td>77-92-9</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>ABTS</td>
			<td>Sigma Aldrich</td>
			<td>30931-67-0</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Sigma Aldrich</td>
			<td>7722-84-1</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>7647-14-5</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 4</td>
			<td>VWR</td>
			<td>332732B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 7</td>
			<td>VWR</td>
			<td>332742D</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer capsules - pH 10</td>
			<td>VWR</td>
			<td>332762H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma Aldrich</td>
			<td>435570</td>
			<td>Irritant. Use in fume cupboard</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>Irritant</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Fisher Scientific</td>
			<td>BFS-525-010A</td>
			<td></td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer +printer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fridge-freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drying apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>+tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flammable liquid storagecabinet</td>
			<td>VWR</td>
			<td>649-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fume cupboard</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot-plate stirrer</td>
			<td>VWR</td>
			<td>640-282</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate washer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Research&#174; pipettes - multipack option 2</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 1000ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 200ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette - 20ul</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette filler</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Progen Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Hilsonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fecal glucocorticoid analysis: non-invasive adrenal monitoring in equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

国内馬（N = 16は、8頭の牝馬、8去勢馬）15年（±3）の平均年齢と性別に応じてグループ化され、社会的孤立のレベルの増加と4住宅のデザインを施した（n = 4の馬/処理）しました。密接に彼らの自然の生息地をシミュレートし、群れ環境に住んでいる住宅1関与馬。屋内納屋でペアに住んでいる住宅2関与馬。ハウジング3関与馬は厩舎ではなく、他の馬と馬のハウジング4?...

Watch this Scientific Journal Video about Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids at JoVE.com

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

fecal-glucocorticoid-analysis-non-invasive-adrenal-monitoring

Research

JoVE Journal

Biology

9.6K ビュー.  Nottingham Trent University. Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

ビデオ: Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

マウス副腎髄質クロマフィン細胞分離

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

シンクロトロンX線トモグラフィを用いたサブミクロン分解能を持つ非侵襲的3次元可視化

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

ゼブラフィッシュ幼虫ルシフェラーゼレポーターシステムを用いたグルココルチコイドシグナル伝達のための化学スクリーニング法

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

誰が誰ですか？非侵襲的な方法に個別にセックス·アンド·ザ·マーク·晩成性の雛

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

LDL コレステロールの取り込みアッセイ セルにライブセル イメージングを使用して正常性の監視

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...