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>
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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 ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

マウス骨髄からの破骨細胞の導出

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

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 ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

リアルタイム細胞代謝フラックス分析装置を使用して骨芽細胞生体エネルギーをモニタリング

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

骨馴化培地：調製およびバイオアッセイ

要約

さらに動画を探す

この動画の章

無細胞培地でボルバキアの維持

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

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

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

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

イメージングのための簡単な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

マウス骨髄からの破骨細胞の導出

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。

ヒト変形性膝関節からの組織採取とRNA抽出

リアルタイム細胞代謝フラックス分析装置を使用して骨芽細胞生体エネルギーをモニタリング

骨馴化培地：調製およびバイオアッセイ

要約

さらに動画を探す

この動画の章

無細胞培地でボルバキアの維持

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

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

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

完全ノックアウト血清代替のフィーダーを含まない培地を用いてヒトiPS細胞のCryopreservingおよびリカバリ

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

マウス骨髄からの破骨細胞の導出

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。

ヒト変形性膝関節からの組織採取とRNA抽出

リアルタイム細胞代謝フラックス分析装置を使用して骨芽細胞生体エネルギーをモニタリング

イメージングのための簡単な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉