改善し、実際の状態にこれらのテクニックを絞り込むに役立つ演習の古いメンバーに感謝したい: 博士シーリア ・ マルティネス-モーラ;博士アンナ Mrowiec、博士カタリナ ルイス自慢、博士アントニア ・ アルカラス ガルシア。これらの技術の開発を強く支援病院臨床ウニベルシタリオ ビルゲン デ ラ Arrixaca にお世話になっております。セルバンテス ・ デ ・またサラッド カルロス 3 世、フォンド ・ デ ・研究 Sanitarias。Estatal を計画私 + D + 私とセルバンテス ・ デ ・ サラッド カルロス III Subdirección 一般 06 y 開発デ ラ危惧 (許可番号: PI13/00794);www.isciii.es. Fondos ・ フェダー「Una manera デ hacer エウロパ」。我々 も行政も支援をグァテマラ ・ デ ・ ムルシア、IMIB Arrixaca、外国金融機関に感謝します。最後に、我々 は博士イサベル ・ マルチネス-Argudo と Facultad デ芸術報 Ambientales y Bioquímica、キャンパス ′ デ ラ Fábrica デ アルマス、グァテマラ カスティーリャ ・ ラ ・ マンチャ州、トレド喜んで譲ることで彼らの親切なサポートのおかげでスペシャルを与えたい、バイオ医薬品と本稿の撮影の一部を実現するためのバイオ テクノロジー研究室。

1. 人工傷スクラッチ アッセイ定量的研究
<ol>
	<li>セル膜の準備
	<ol>
		<li>無菌状態の下で働いて、シードし、血清添加培地を用いた培養フラスコに Mv1Lu または HaCaT 上皮細胞を成長します。一度、すべての 24-48 h 媒体を更新します。電池が 80% の合流点に達する後、適切な方法、すなわちtrypsinization9を使用してセルをデタッチします。 注: Mv1Lu と実装さ?...

<ol>
	<li>Singer, A. J., Clark, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+wound+healing.">Cutaneous wound healing.</a> N Engl J Med. 341 (10), 738-746 (1999).</li><li>Davidson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+wound+repair.">Animal models for wound repair.</a> Arch Dermatol Res. 290, S1-S11 (1998).</li><li>Alcaraz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+Membrane+Modifies+the+Genetic+Program+Induced+by+TGFss,+Stimulating+Keratinocyte+Proliferation+and+Migration+in+Chronic+Wounds.">Amniotic Membrane Modifies the Genetic Program Induced by TGFss, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds.</a> PLoS One. 10 (8), e0135324 (2015).</li><li>Ruiz-Canada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+stimulates+cell+migration+by+modulating+Transforming+Growth+Factor-beta+signaling.">Amniotic membrane stimulates cell migration by modulating Transforming Growth Factor-beta signaling.</a> J Tissue Eng Regen Med. , (2017).</li><li>Schmierer, B., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta-SMAD+signal+transduction:+molecular+specificity+and+functional+flexibility.">TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility.</a> Nat Rev Mol Cell Biol. 8 (12), 970-982 (2007).</li><li>Hu, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAK+and+paxillin+dynamics+at+focal+adhesions+in+the+protrusions+of+migrating+cells.">FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells.</a> Sci Rep. 4, 6024 (2014).</li><li>Martinez-Mora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroin+and+sericin+from+Bombyx+mori+silk+stimulate+cell+migration+through+upregulation+and+phosphorylation+of+c-Jun.">Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.</a> PloS one. 7 (7), e42271 (2012).</li><li>Bernabe-Garcia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oleanolic+acid+induces+migration+in+Mv1Lu+and+MDA-MB-231+epithelial+cells+involving+EGF+receptor+and+MAP+kinases+activation.">Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation.</a> PLoS One. 12 (2), e0172574 (2017).</li><li>Huang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J Biomed Sci. 17, 36 (2010).</li><li>Liberio, M. S., Sadowski, M. C., Soekmadji, C., Davis, R. A., Nelson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+tissue+culture+coating+substrates+on+prostate+cancer+cell+adherence,+morphology+and+behavior.">Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior.</a> PLoS One. 9 (11), e112122 (2014).</li><li>Pierreux, C. E., Nicolas, F. J., Hill, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta-independent+shuttling+of+Smad4+between+the+cytoplasm+and+nucleus.">Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.</a> Mol Cell Biol. 20 (23), 9041-9054 (2000).</li><li>Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cytokines+in+wound+healing.">Growth factors and cytokines in wound healing.</a> Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16 (5), 585-601 (2008).</li><li>Ansell, D. M., Holden, K. A., Hardman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+repair:+Are+they+cutting+it?.">Animal models of wound repair: Are they cutting it?.</a> Exp Dermatol. 21 (8), 581-585 (2012).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453 (7193), 314-321 (2008).</li><li>Insausti, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+induces+epithelialization+in+massive+posttraumatic+wounds.">Amniotic membrane induces epithelialization in massive posttraumatic wounds.</a> Wound Repair Regen. 18 (4), 368-377 (2010).</li><li>Wu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+arrest+in+G0/G1+phase+by+contact+inhibition+and+TGF-beta+1+in+mink+Mv1Lu+lung+epithelial+cells.">Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells.</a> Am J Physiol. 270 (5 Pt 1), L879-L888 (1996).</li><li>Boukamp, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Marshall, J., Wells, C. M., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Szybalski, W., Iyer, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+DNA+by+Enzymatically+or+Chemically+Activated+Mitomycins+and+Porfiromycins,+Bifunctionally+"Alkylating"+Antibiotics.">Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics.</a> Fed Proc. 23, 946-957 (1964).</li><li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C:+small,+fast+and+deadly+(but+very+selective).">Mitomycin C: small, fast and deadly (but very selective).</a> Chem Biol. 2 (9), 575-579 (1995).</li><li>Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+using+silicone+elastomer+masks+for+quantitative+analysis+of+cell+migration+without+cellular+damage+or+substrate+disruption.">A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.</a> Cell Adh Migr. 7 (6), 469-475 (2013).</li><li>Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+in+generating+lipid+second+messengers+in+signal+transduction.">Role of phospholipase in generating lipid second messengers in signal transduction.</a> FASEB J. 5 (7), 2068-2077 (1991).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li></ol>

皮膚または粘膜の破壊、線維芽細胞、上皮や免疫細胞など、様々 な細胞タイプの行動によってバリア機能が復元されます。相まって、これらの細胞を受ける複雑なアポトーシス、増殖、分化、重要なは、線維芽細胞を含むプロセスと上皮細胞の移行、混乱の組織の修復のための責任の究極のメカニズムであると表在性上皮ギャップ1,12細胞遊走を勉...

上皮のギャップの最終的な閉鎖と混乱の表面1の修復を担当しており、細胞遊走は創傷治癒に必要です。動物モデルにおける人工傷を実行できるように生理的条件2の近くでこの複雑なプロセスを複製するため。しかし、このアプローチは多くの場合コストがかかり、複雑な実験の手順、可能性のある創傷治癒過程の複雑な性質のための明確なプロセスの研究の精度を欠いている結果します。
上皮細胞の体外培養は、創傷治癒や処理セルの回遊行動に及ぼす影響のこれらの細胞が果たす役割を研究するため動物モデルの有用な代替を提供します。上皮細胞の生理学は非合流文化3,4,5,6; を使用して分子技術によってよく勉強しました。ただし、上皮整合性の破壊は結構機械的切開により通常。細胞培養、細胞のほとんどの数は、傷のギャップにさらされるかもしれないし、彼らは分子生物学の技術のための余りに小さいサンプルを表すこれを意味します。ただし、これらの病変は、ミンク肺上皮細胞 (Mv1Lu) または自然不死化ひと表皮細胞 (HaCaT) セルなど、いくつかの上皮細胞ラインの生得的な渡り鳥の特性を利用して、顕微鏡のスケールで学ぶことができます。ライン。
ここでは創傷治癒の3,4,7,8のコンテキストにおける上皮細胞の移行に関する定量的データを取得する適切な顕微鏡法について述べる。また、移行上皮膜で発生した質的分子および形態学的変化を研究に役立つその他の方法を提案する.全体的にみて、これらのメソッドは、創傷治癒過程におけるダイナミクスと上皮細胞の挙動と治療への応答に関与の形態的変化を研究するためのフレームワークを提供します。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Biowest, Nuaill&#233;, France</td>
			<td>L0102-500</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>Eagles&#8217;s Minimum Essential Medium (EMEM)</td>
			<td>Lonza</td>
			<td>BE12 -662F</td>
			<td>Optional 10 % FBS supplement</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td>Use at 2 mM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA USA</td>
			<td>DE17603A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T4049</td>
			<td>Dilute as appropriate</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9155</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS) (10x)</td>
			<td>Gibco by Life Technologies</td>
			<td>14200-067</td>
			<td>Dilute to 1x</td>
		</tr>
		<tr>
			<td>24-well culture plates</td>
			<td>BD FALCON//SARSTED</td>
			<td>734-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>SARSTEDT</td>
			<td>83-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>E9644</td>
			<td>Used 10 ng/mL</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>MENZEL-GL&#196;SER</td>
			<td>MENZCB00120RA020</td>
			<td>SHORT DEPTH OF FIELD</td>
		</tr>
		<tr>
			<td>Reinforced razor blade no. 743</td>
			<td>Martor (through VWR)</td>
			<td>MARO743.50</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0541</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l sterile aerosol pipet tips</td>
			<td>VWR</td>
			<td>732-0528</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera coupled phase contrast microscope</td>
			<td>Motic Spain</td>
			<td>Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>LSM 510 META</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Culture dish</td>
			<td>BD FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti c-Jun antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-1694</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG (polyclonal goat ) AF 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Used 1:400</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>14530</td>
			<td>Used 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (in methanol) (red)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>Used 1:100</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>BD DIFCO</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health, USA</td>
			<td>Release 1.50i</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen LSM 510 image processing software</td>
			<td>ZEISS Microimaging, Germany</td>
			<td>Release 5.0 SP 1.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy based methods for the assessment of epithelial cell migration during in vitro wound healing

細胞遊走は、創傷治癒に必須の側面です。人工を作成する傷を研究の動物モデルにコストがかかり、複雑な実験手順の結果にしばしば潜在的精度に欠けている間。上皮細胞の体外培養は、創傷治癒と細胞の治療法の影響で細胞の回遊行動の研究のため適切なプラットフォームを提供します。上皮細胞の生理学は非合流条件でよく勉強しました。ただし、このアプローチは、自然治癒で条件に類似しないかもしれない。機械的手段によって上皮の整合性を中断することは現実的なモデルを生成しますが、分子技術のアプリケーションを妨げることがあります。したがって、顕微鏡技術は上皮細胞の遊走を勉強に最適体外。ここで我々 は, 上皮細胞の渡り鳥の性能に関する量的・質的データを入手できる人工移行フロント アッセイ、人工傷スクラッチ試験 2 つの特定の方法を詳しく説明します。

定量的研究のため人工傷スクラッチ試金: 移行上皮成長因子 (EGF) 推進を評価します。
EGF は、上皮細胞の増殖と移行、およびこうして移行推進を定量化するための肯定的な制御のよく知られている誘導です。Mv1Lu と HaCaT 細胞膜が傷傷試金で使用された、治療前の写真が得られました。10 ng/ml の EGF, 接種後のセルは固定と治療...

Watch this Scientific Journal Video about Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing at JoVE.com

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

本稿では培養上皮細胞は、どのように切開のような病変便利なモデルの傷癒しの in vitro、共焦点イメージングを可能にするについて説明しますまたはレーザ走査顕微鏡と高品質を提供することができます。細胞の挙動と移行に関与するメカニズムの両方の研究のための質的・量的データ。

顕微鏡法によるIn Vitro創傷が治癒過程における上皮移行の評価

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

The overall goal of these epithelial cell culture artificial wounding procedures is to study cell migration from both a quantitative and qualitive perspective, allowing assessment of the effects of specific treatments of interest on wound healing. This method can help answer key questions in the wound-healing field, as they allow the precise characterization of the roles of specific migration processes during epithelial disruption. The main advantage of this approach is that it allows the correlation of cell migratory measurements with changes in the behavior of relevant molecular markers.Implication of these techniques extend towards a therapy of clinical wounds, as it offers a convenient setup for early drug testing and for wound closure. Generally in to this method will start because of difficulties in obtaining consistent reproduceability in the wounds with an generation. For an artificial wound scratch assay, begin by seeding each well of a culture plate with two milliliters of epithelial cells of interest in complete medium.Enculture the cells at 37 degrees Celsius and five percent carbon dioxide for two to three days until 100%confluency. When the cells are ready, wash the cells two times with fresh serum-free medium followed by 24 hours of culture in fresh serum-free medium. The next day, place the plate in a sterile work area and drag the narrow end of a sterile micro-pipette tip across the bottom of each well two times to generate a cross-shaped wound.A consistent wound gap is achieved by applying constant, firm pressure while smoothly moving the tip along the epithelial surface. Excessive pressure will deform the tip, causing an irregular wound radius. Gently remove the supernatant to discard the detached cells and carefully add fresh serum-free medium to the wells.Using in inverted phase contrast microscope connected to a charge coupled device camera, align the edge of the microscope field with the adjacent intersection of the scratches to obtain up to four pre-treatment reference images of the scratch gap in each well. When all of the images have been acquired, replace the medium in the designated treatment wells with fresh medium, supplemented with selected treatments of interest and return the plates to the cell culture incubator. When at least one condition reaches about 90%scratch gap closure, gently replace the supernatant with one milliliter of four percent formalin in PBS for a 15 minute incubation at room temperature.Then gently wash the cells with PBS at least three times to remove any excess formalin and image the wells as just demonstrated, using the same imaging parameters in the original reference areas captured after scratching. To analyze the images using the appropriate image processing software, open the recorded, pre-treatment image of interest and under the Image menu, set the image type to 8-bit. Under the Process menu, select the Filters sub-menu and apply Variance filtering.Under the Image menu, open the Adjust sub-menu and set the Threshold to black and white. In the Process menu, select the Binary sub-menu and apply Fill Holes. Under the Analyze menu, select Set Measurements and activate Area.Then draw the measurable area, following the migrating edge contour to delimit the gap. Under the Analyze menu, select Analyze Particles and record the total area values for the drawn area surface. To quantify the absolute migration for each set of individual samples as the difference between the gap surface measurements, export the pre and post-treatment total gap surface area values to a spreadsheet and plot the quantification outputs.For an artificial migration front assay, under sterile conditions, add one layer of up to 33 sterilized round coverslips into empty 10 centimeter culture plates. When the plate surface is completely covered, gently seed the epithelial cells of interest at a concentration that allows for 100%confluence after two or three days of culture. If necessary, gently depress the coversips with a sterile micropipette tip to prevent floating.When the cells reach confluency, replace the supernatant with serum-free medium for another 24 hours of culture. Then, use sterile tweesers to carefully transfer one coverslip into a new 10 centimeter plate containing fresh serum-free medium, taking care to hold the coverslip along its periphery. To create the artificial wounds, drag a sterilized razor blade back and forth across each coverslip to make a three to four millimeter transverse line over the center of each cell culture to completely remove the central monolayer strip.Take care to place the razor blade edge heavily across the coverslip surface when making the wound to safeguard against an irregular edge shape and the generation of flopping epithelial portions. Transfer up to four wound coverslips, cell side up, into individual wells of a six well plate, containing at least two milliliters of serum-free medium and gently wash each well two times with fresh serum-free medium to remove any detached cells. When all of the detached cells have been discarded, gently replace the wash medium with fresh medium supplemented with the treatments of interest, and place the plate in the cell culture incubator.At the end of the appropriate experimental period, fix the cells with four percent formalin in PBS as just demonstrated, and stain the cells with the appropriate fluorescent conjugated antibodies of interest. Then image the structural changes occurring at the migrating front edge by fluorescence microscopy according to the experimental parameters of interest. In this experiment, the wound gaps were measured before and after treatment with epidermal growth factor, a well-known inducer of epithelial cell proliferation and migration.The absolute migration was then calculated as the difference between the pre and post-treatment gap surface areas. Treatment with epidermal growth factor potentiates cell cytoskeletal dynamics as observed in these laser scanning microscopy images of F-Actin staining of control and growth factor stimulated cells. Further, c-Jun over-expression is observed with an increased expression of the protein apparent in the cells adjacent to the migrating front.While attempting this procedure, it's important to remember to check the integrity of the wound edges for the presence of epithelial cell flaps that may disrupt the migration quantification. After watching this video, you should have a good understanding of how to both quantitatively and qualitatively assess the migratory behavior of the

Research

JoVE Journal

Medicine

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

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医学

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Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

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準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

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