Authors would like to thank Catherine Solioz for her skillful assistance.

1. BCMの準備<ol><li>できるだけ新鮮な地元の肉屋から豚の下顎を取得します。しっかりと表面に下顎を置き、骨に接続されているすべての軟組織や骨膜を残さない特別な注意を払って全厚フラップをリリース。流フードの下で働くために必要とすることなく、クリーンな環境で動作します。 </li><li>全層フラップが解放されると、頬側から骨片を採取するために、骨のスクレーパーを使用しています。骨スクレーパーがシャープにする必要がありますのでご注意ください。しっかりと骨のスクレーパーを処理し、長い運動が骨を収集しています。小さいその1ミリメートルの骨チップを捨てます。 <ol><li>ネイティブの骨チップを維持するために、1％抗生物質およびそれらを乾燥させない抗真菌剤を添加したダルベッコ改変イーグル培地（DMEM）で直径10cmのプラスチック皿に直接骨チップを配置します。 </li><li>熱処理の影響を評価するために、被験者の骨チップは、80℃で30分間低温殺菌するか、または121℃で20分間オ​​ートクレーブ。 </li><li>脱塩の影響を評価するために、4℃で4~6時間、1MのHCLに骨チップを振るし、pHが中性になるまで培養液で繰り返し洗浄します。 </li></ol></li><li> 10ミリリットルあたりの骨チップ5gの新しいプラスチック皿に1％の抗生物質および抗真菌剤を補充した新鮮なDMEMの合計を配置します。 </li><li> 24時間、37℃の加湿雰囲気中で、プラスチック皿を置きます。そして、収穫BCM。遠心分離機、残骸を削除する滅菌（0.2 nm）をフィルタリングし、-80℃で凍結アリコートを維持するために10分間、200×gでBCM。 </li><li>使用直前にBCMストックを解凍し、凍結融解の繰り返しサイクルを避けます。 </li><li>示された実験のために、RTで1時間BCMまたは無血清培地を用いてコラーゲン膜を浸します。 PBSと激しく膜を洗浄し、96ウェルプレートに入れます。湿潤膜は、細胞を播種します。 </li><li> BCMの調製方法を図1に要約されています。 </LI&gt; </ol>間葉系細胞に基づく2バイオアッセイ<ol><li> 30,000細胞/ cm 2の濃度で12ウェルプレートにシード（例えば、骨細胞、歯肉および歯根膜線維芽細胞のための）ヒト間葉系細胞。細胞を播種するにはDMEM、10％ウシ胎児血清および抗生物質からなる成長培地を使用します。細胞がプレートO / Nに接続してみましょう。 </li><li>培地を廃棄し、37℃で予め加温したPBSで細胞を洗浄します。で、20％BCMなし予め温めた無血清培地を添加することによって細胞を刺激します。 24時間、37℃で加湿雰囲気中で細胞を配置します。 </li><li> 、培地を捨て、予め温めておいたPBSで細胞を洗浄し、お好みのプロトコルに従ってRNAを抽出します。 </li><li>各試料中のRNAの同量を有するためにRNAの濃度を調整します。 cDNAを準備し、 表1に示すプライマーを用いて、選択された遺伝子を分析するためのqRT-PCRを行います。 注：2×SYBRグリーン、前方の20xプライマー、20Xプライマーリバース、5倍の滅菌DD水、5倍のcDNA：これらは、すべてのコンポーネントの希釈液です。定量RT-PCRは、95℃15秒、60℃1分の40サイクルで行われます。 </li><li> CT GAPDHおよびΔ（ΔCT）ΔCT刺激される - - ΔCT制御ΔCTがCTターゲットであるΔ（ΔCtの）メソッドを使用して、ハウスキーピング遺伝子GAPDHに正規化することによって、相対的な発現レベルを計算します。 </li><li>この品質管理の後、間葉細胞、造血細胞または内皮細胞を含む細胞の全ての種類を刺激するために培地にBCMを追加します。 </li></ol>

<ol>
	<li>Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Stability+of+Early+Implant+Placement+with+Contour+Augmentation.">Long-term Stability of Early Implant Placement with Contour Augmentation.</a> Journal of dental research. , (2013).</li><li>Chiapasco, M., Casentini, P., Bone Zaniboni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=augmentation+procedures+in+implant+dentistry.">augmentation procedures in implant dentistry.</a> The International journal of oral &amp; maxillofacial implants. 24 Suppl. , 237-259 (2009).</li><li>Giannoudis, P. V., Dinopoulos, H., Bone Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=substitutes:+an+update.+Injury.">substitutes: an update. Injury.</a> 36, Suppl 3. S20-S27. , (2005).</li><li>Jensen, S. S., Broggini, N., Hjorting-Hansen, E., Schenk, R., Bone Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=healing+and+graft+resorption+of+autograft,+anorganic+bovine+bone+and+beta-tricalcium+phosphate.+A+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 17, 237-243 (2006).</li><li>Jensen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+biphasic+calcium+phosphate+in+standardized+bone+defects:+a+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 18, 752-760 (2007).</li><li>Grabowski, G., Bone Cornett, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=graft+and+bone+graft+substitutes+in+spine+surgery:+current+concepts+and+controversies.">graft and bone graft substitutes in spine surgery: current concepts and controversies.</a> The Journal of the American Academy of Orthopaedic Surgeons. 21, 51-60 (2013).</li><li>Khan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+bone+grafting.">The biology of bone grafting.</a> The Journal of the American Academy of Orthopaedic Surgeons. 13, 77-86 (2005).</li><li>Bohr, H., Ravn, H. O., Werner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteogenic+effect+of+bone+transplants+in+rabbits.+The+Journal+of+bone+and+joint+surgery.">The osteogenic effect of bone transplants in rabbits. The Journal of bone and joint surgery.</a> British. 50, 866-873 (1968).</li><li>Bone Urist, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=formation+by+autoinduction.">formation by autoinduction.</a> Science. 150, 893-899 (1965).</li><li>Caball&#233;-Serrano, J. D., Buser, D., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+porcine+bone+conditioned+medium.">Proteomic analysis of porcine bone conditioned medium.</a> The International journal of oral &amp; maxillofacial implants. , (2014).</li><li>Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-Conditioned+Medium+Inhibits+Osteogenic+and+Adipogenic+Differentiation+of+Mesenchymal+Cells+In+Vitro.">Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro.</a> Clinical implant dentistry and related research. , (2014).</li><li>Brolese, E., Buser, D., Kuchler, U., Schaller, B., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+chips+release+of+sclerostin+and+FGF-23+into+the+culture+medium:+an+in+vitro+pilot+study.">Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study.</a> Clinical oral implants research. , (2014).</li><li>Caball&#233;-Serrano, J., Bosshardt, D. D., Gargallo-Albiol, J., Buser, D., Bone Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=conditioned+medium+enhances+osteoclastogenesis+in+murine+bone+marrow+cultures.+Clin+Oral+Impl+Res;+in.">conditioned medium enhances osteoclastogenesis in murine bone marrow cultures. Clin Oral Impl Res; in.</a> Int J Oral Maxillofac Surg. , (2015).</li><li>Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-conditioned+medium+changes+gene+expression+in+bone-derived+fibroblasts).">Bone-conditioned medium changes gene expression in bone-derived fibroblasts).</a> IJOMI accepted. , (2014).</li><li>Fulzele, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelopoiesis+is+regulated+by+osteocytes+through+Gsalpha-dependent+signaling.">Myelopoiesis is regulated by osteocytes through Gsalpha-dependent signaling.</a> Blood. 121, 930-939 (2013).</li><li>Becerra, J., Andrades, J. A., Ertl, D. C., Sorgente, N., Nimni, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demineralized+bone+matrix+mediates+differentiation+of+bone+marrow+stromal+cells+in+vitro:+effect+of+age+of+cell+donor.">Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.</a> Journal of. 11, 1703-1714 (1996).</li><li>Kupcova Skalnikova, ., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+techniques+for+characterisation+of+mesenchymal+stem+cell+secretome.">Proteomic techniques for characterisation of mesenchymal stem cell secretome.</a> Biochimie. , (2013).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cell+secretome:+A+novel+role+for+progranulin+during+risedronate+treatment.">Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment.</a> , (2013).</li><li>Schuldt Filho, ., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+of+demineralized+bone+matrix+activates+TGF-&#946;+signaling+pathways+in+mesenchymal+cells+in+vitro.">Conditioned medium of demineralized bone matrix activates TGF-&#946; signaling pathways in mesenchymal cells in vitro.</a> J Cranio Maxill Surg. , (2015).</li><li>Buser, D., Chen, S. T., Weber, H. P., Belser, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+implant+placement+following+single-tooth+extraction+in+the+esthetic+zone:+biologic+rationale+and+surgical+procedures).">Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures).</a> The International journal of periodontics &amp; restorative dentistry. 28, 441-451 (2008).</li><li>Stoecklin-Wasmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbable+collagen+membranes+for+periodontal+regeneration:+a+systematic+review.">Absorbable collagen membranes for periodontal regeneration: a systematic review.</a> Journal of dental research. 92, 773-781 (2013).</li></ol>

著者らは、開示することは何もありません。ジョルディカバリエ·セラーノは、歯科研究と教育、バーゼル、スイスの財団から奨学金を受けました。

骨馴化培地は、骨再生の初期段階の間に骨移植片の解放の活動を反映しています。ここで説明するプロトコルは、骨再生に関与する細胞の異なるタイプの応答を研究するために適合させることができます。さらに、プロトコルは、処理骨または骨充填剤からの馴化培地を調製するために用いることができます。様々な天然および処理骨から放出因子：方法は実行し、シンプルなコンセプトに?...

自家骨は広く奇形、resective手術、再建外傷外科手術、および配置1,2を注入する前の結果として発生した欠陥を埋めるために使用されます。骨移植片は、移植片の統合のプロセスをサポートする方法の生物学的な原理を理解することは自家移植が再建手術におけるゴールドスタンダードであると考えられている理由を理解するための唯一のキーではない、それはまた、代用骨3の改良されたデザインにバイオニックです。それでも、移植片の統合は、より高速な自家骨と骨置換4,5に比べてです。これにより、骨再生をサポートするので、効果的な自家骨を作る分子および細胞のメカニズムを明らかにすることが不可欠です。 統合プロセス6,7を支持すると考えられる自家移植片の三教科書特性があります。まず、自家骨が成長するために、新たに形成された骨のためのガイダンスを提供し、骨伝導性であり、欠陥へ。第二に、自家骨は、骨芽細胞に分化することができる8間葉細胞を含むことを意味し、骨形成性です。第三に、自家骨は、マトリクス状に埋葬骨形成タンパク質のような成長因子は、軟骨内、あるいは膜内骨形成9のプロセスを開始することができるように骨誘導性です。別の側面があります：新たに調製した骨片は「骨馴化培地」10〜15 を用いたin vitroの観察に基づいて、パラクリン機能を保持します。また、骨髄造血の影響は、16を言及すべきです。類似の用語「脱灰骨基質馴化培地は、「すでに脱塩17によって処理される場合であっても、1996年に造語と骨のパラクリン機能の全体的な概念をサポートしていました。我々の目的のために、BCMは10,11を下顎骨新鮮なブタから調製することができます。 BCMのプロテオーム解析は、成長事実を含め、複雑な組成を明らかにしましたORSと細胞外マトリックス10の構成要素は、また、全体の骨18,19のプロテアソームの既存の知識を拡張します。このように、BCMは、in vitroで骨移植片の様々な改変の解放の活動を反映する必要があります。 間葉系細胞は、例えば骨チップから、または口腔の軟組織から単離されたもののため、BCMする？ インビトロで露出しているときに何が起こるか、BCMは、骨形成および脂肪生成分化を減少させ、IL11発現11の強力な増加を引き起こします。全ゲノムマイクロアレイは示差BCMに応答して、間葉系細胞で発現する複数の遺伝子を明らかにしました。これらの遺伝子の中アドレノメデュリン（ADM）、IL11、IL33、NADPHオキシダーゼ4（のNox4）、プロテオグリカン4（PRG4、またはルブリシン）とペントラキシン3（PTX3）15があります。オートクレーブ処理骨チップから得られたBCMは、それぞれの遺伝子14の発現を変更することができませんでした。低温殺菌および凍結を受けた骨チップからBCMをすることができました遺伝子発現14を変更します。脱灰骨基質（DBM-CM）のも馴化培地は、TGF-β調節遺伝子20の発現を変化させます。興味深いことに、周囲の軟組織21,22の骨チップを保護するために使用されるコラーゲンバリア膜は、遺伝子発現の23の変化の原因であるBCMの部分を吸着させました。 BCMの研究がいくつか例を挙げると、例えば、骨吸収破骨細胞および内皮細胞のような骨再生に関与する他の細胞型に拡張することができます。全体的に、蓄積インビトロのデータは、前臨床試験の設計のための科学的根拠を提供します。 本プロトコルは、2つあります：まず、BCMを準備する方法を示しています。第二に、in vitroでの間葉系細胞に基づいて、その生物学的活性をテストする方法を示しています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pig Mandibles</td>
			<td>Local bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Scraper</td>
			<td>Hu-Friedy</td>
			<td>PPBUSE2/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics &#38; Antimicotics</td>
			<td>All life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Membranes (Bio-Gide)</td>
			<td>Geistlich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Invitrogen Corporation</td>
			<td align="right">16030074</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Corporation</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			High Pure RNA Isolation Kit</td>
			<td>Roche</td>
			<td align="right">11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche</td>
			<td align="right">4379012001</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Microsynth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (for Q-RT-PCR)</td>
			<td>Roche</td>
			<td align="right">4673484001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td align="right">11666789001</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone conditioned medium: preparation and bioassay

自家骨移植が広く口腔外科、整形外科、および外傷学で使用されています。自家骨移植が唯一欠けている骨を交換しないで、彼らはまた、骨再生の複雑なプロセスをサポートしています。骨伝導、骨形成、および骨誘導性：自家移植のこの有利な動作は、次の3つの特性に起因します。しかし、他の態様が存在する：骨は、骨再生に関与する間葉細胞を標的とすることができる成長因子、を含む分子、無数の移植片を解放します。骨移植片のパラクリン特性は、骨馴化培地（BCM）を使用してインビトロで研究することができます。ここでは、熱処理または脱塩を受けたネイティブ豚の皮質骨から骨馴化培地を調製する方法に関するプロトコル、および骨を提示します。細胞は、直接BCMにさらされたり、コラーゲン膜、以前BCMに浸したような生体材料上に播種することができます。我々は、in vitroで bioassaの例を与えますTGF-β調節遺伝子の発現に、間葉系細胞とYS。提示されたプロトコルは、さらに、骨再生の際に骨移植片のパラクリン効果を明らかにし、再建手術の幅広い分野でトランスレーショナルリサーチのためのパスを開くことが奨励すべきです。

骨馴化培地を、新鮮なブタの骨片から製造されます。 BCMを準備し、BCMと組み合わせて生体材料を使用するプロセスの概要をそれぞれ図1および図2に示されています。 BCMの準備中に、最終的なBCMの品質に影響を与えることができ、短い運動または非常に小さな骨チップ限り動きに大きな骨チップを得ることが重要です。 ADM、PTX3、IL11、IL33、PRG4のNox4と（ 図3）：B...

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Bone Conditioned Medium: Preparation and Bioassay

我々は、骨馴化培地（BCM）を調製し、インビトロでその活性をテストする方法をここで説明します。

骨馴化培地：調製およびバイオアッセイ

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

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JoVE Journal

Biology

11.0K ビュー.  School of Dental Medicine, University of Bern. 我々は、骨馴化培地（BCM）を調製し、インビトロでその活性をテストする方法をここで説明します。 

ビデオ: Bone Conditioned Medium: Preparation and Bioassay

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

無細胞培地でボルバキアの維持

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

侵害受容性および炎症性メディエーターの組織特異的な測定のためのヒトのin vivoバイオアッセイ

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

組織学、組織形態計測および蛍光色素分析のためのUndecalcified骨の準備

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

宿主植物の揮発性物質のスクリーニングツールとしてElectroantennographicバイオアッセイ

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

フローサイトメトリー法による細胞小胞細胞調節されたメディアからの解析します。

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

ヒト変形性膝関節からの組織採取とRNA抽出

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

蚊やショウジョウバエに対する殺虫剤毒性を定量するための局所適用バイオアッセイ

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

食品鉄バイオアベイラビリティ測定のためのCaco-2細胞バイオアッセイ

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...