Name: imaging molecular adhesion in cell rolling by adhesion footprint assay
Uploaded: 2021-09-27T15:00:00
Description: Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

この研究は、カナダイノベーション財団(CFI 35492)、カナダディスカバリーグラント自然科学工学研究評議会(RGPIN-2017-04407)、研究基金のニューフロンティア(NFRFE-2018-00969)、マイケル・スミス健康研究財団(SCH-2020-0559)、ブリティッシュコロンビア大学エミンズ基金によって支援されました。

1. オリゴヌクレオチド標識とハイブリダイゼーション
<ol><li>プロテインGジスルフィド結合の還元<ol>
<li>1mLの超純水にプロテインG(ProtG)10mgを溶かします。 注:ここでのプロテインGは、C末端の単一のシステイン残基とN末端ポリヒスチジンタグで修飾されています。</li>
		<li>バッファー交換≥20 μL ProtG (10 mg/mL) を P6 カラムを使用して 1x PBS (pH 7.2) に交換します。</li>
		<li>バッファ...

<ol>
	<li>McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+cell+adhesion.">Rolling cell adhesion.</a> Annual Review of Cell and Developmental Biology. 26 (1), 363-396 (2010).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+The+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: The leukocyte adhesion cascade updated.</a> Nature Reviews Immunology. 7 (9), 678-689 (2007).</li><li>Zarbock, A., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesion+and+activation+under+flow.">Neutrophil adhesion and activation under flow.</a> Microcirculation. 16 (1), 31-42 (2009).</li><li>Etzioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+molecules+-+Their+role+in+health+and+disease.">Adhesion molecules - Their role in health and disease.</a> Pediatric Research. 39 (2), 191-198 (1996).</li><li>van de Vijver, E., vanden Berg, T. K., Kuijpers, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+adhesion+deficiencies.">Leukocyte adhesion deficiencies.</a> Hematology/Oncology Clinics of North America. 27 (1), 101-116 (2013).</li><li>Hanna, S., Etzioni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+adhesion+deficiencies.">Leukocyte adhesion deficiencies.</a> Annals of the New York Academy of Sciences. 1250 (1), 50-55 (2012).</li><li>Geng, Y., Marshall, J. R., King, 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=Glycomechanics+of+the+metastatic+cascade:+Tumor+cell-endothelial+cell+interactions+in+the+circulation.">Glycomechanics of the metastatic cascade: Tumor cell-endothelial cell interactions in the circulation.</a> Annals of Biomedical Engineering. 40 (4), 790-805 (2012).</li><li>Yasmin-Karim, S., King, M. R., Messing, E. M., Lee, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-selectin+ligand-1+controls+circulating+prostate+cancer+cell+rolling/adhesion+and+metastasis.">E-selectin ligand-1 controls circulating prostate cancer cell rolling/adhesion and metastasis.</a> Oncotarget. 5 (23), 12097-12110 (2014).</li><li>Marshall, B. T., Long, M., Piper, J. W., Yago, T., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+catch+bonds+involving+cell-adhesion+molecules.">Direct observation of catch bonds involving cell-adhesion molecules.</a> Nature. 423 (6936), 190-193 (2003).</li><li>Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+dynamics.">Adhesive dynamics.</a> Journal of Biomechanical Engineering. 136 (2), 021006 (2014).</li><li>Yasunaga, A. B., Murad, Y., Kapras, V., Menard, F., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+interpretation+of+cell+rolling+velocity+distribution.">Quantitative interpretation of cell rolling velocity distribution.</a> Biophysical Journal. 120 (12), 2511-2520 (2021).</li><li>Li, I. T. S., Ha, T., Chemla, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cell+surface+adhesion+by+rotation+tracking+and+adhesion+footprinting.">Mapping cell surface adhesion by rotation tracking and adhesion footprinting.</a> Scientific Reports. 7 (1), 44502 (2017).</li><li>Yasunaga, A. B., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+fast+molecular+adhesion+by+fluorescence+footprinting.">Quantification of fast molecular adhesion by fluorescence footprinting.</a> Science Advances. 7 (34), (2021).</li><li>Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M., Zhuang, X. <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+fluorophores+for+optimal+performance+in+localization-based+super-resolution+imaging.">Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.</a> Nature Methods. 8 (12), 1027-1040 (2011).</li><li>Zhu, M., Lerum, M. Z., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+prepare+reproducible,+homogeneous,+and+hydrolytically+stable+aminosilane-derived+layers+on+silica.">How to prepare reproducible, homogeneous, and hydrolytically stable aminosilane-derived layers on silica.</a> Langmuir. 28 (1), 416-423 (2012).</li><li>Chandradoss, S. D., Haagsma, A. C., Lee, Y. K., Hwang, J. H., Nam, J. M., Joo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> Journal of Visualized Experiments: JoVE. (86), e50549 (2014).</li><li>Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., Jungmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+with+DNA-PAINT.">Super-resolution microscopy with DNA-PAINT.</a> Nature Protocols. 12 (6), 1198-1228 (2017).</li><li>Chalfoun, J., 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=MIST:+Accurate+and+scalable+microscopy+image+stitching+tool+with+stage+modeling+and+error+minimization.">MIST: Accurate and scalable microscopy image stitching tool with stage modeling and error minimization.</a> Scientific Reports. 7 (1), 4988 (2017).</li><li>Wang, X., 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=Constructing+modular+and+universal+single+molecule+tension+sensor+using+protein+G+to+study+mechano-sensitive+receptors.">Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors.</a> Scientific Reports. 6, 1-10 (2016).</li><li>Crockett-Torabi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectins+and+mechanisms+of+signal+transduction.">Selectins and mechanisms of signal transduction.</a> Journal of Leukocyte Biology. 63 (1), 1-14 (1998).</li><li>Ye, Z., 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=The+P-selectin+and+PSGL-1+axis+accelerates+atherosclerosis+via+activation+of+dendritic+cells+by+the+TLR4+signaling+pathway.">The P-selectin and PSGL-1 axis accelerates atherosclerosis via activation of dendritic cells by the TLR4 signaling pathway.</a> Cell Death and Disease. 10 (7), 507 (2019).</li><li>Saha, K., Bender, F., Gizeli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+IgG+binding+to+proteins+G+and+A:+Nonequilibrium+kinetic+and+binding+constant+determination+with+the+acoustic+waveguide+device.">Comparative study of IgG binding to proteins G and A: Nonequilibrium kinetic and binding constant determination with the acoustic waveguide device.</a> Analytical Chemistry. 75 (4), 835-842 (2003).</li><li>Murad, Y., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+molecular+forces+with+serially+connected+force+sensors.">Quantifying molecular forces with serially connected force sensors.</a> Biophysical Journal. 116 (7), 1282-1291 (2019).</li><li>Yasunaga, A. B., Murad, Y., Li, I. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+molecular+tension-classifications,+interpretations+and+limitations+of+force+sensors.">Quantifying molecular tension-classifications, interpretations and limitations of force sensors.</a> Physical Biology. 17 (1), 011001 (2020).</li></ol>

接着フットプリントアッセイにより、細胞圧延接着中のPSGL-1とP-セレクチン間の分子接着事象の可視化が可能になります。このプロセスは、P-selectin媒介捕捉によって開始され、その後、流体せん断応力下での転がりが続きます。実験中の潜在的な問題は、通常、細胞がうまく転がっていても、細胞の転がりが悪かったり、蛍光トラックが欠落したりする。これらの問題は、多くの場合、プロ?...

転がり接着カスケードは、循環細胞が血管壁1に沿ってどのようにテザーおよび転がするかを記述する。受動圧延は主に細胞接着分子(CAM)1の主要クラスであるセレクチンによって媒介される。血液のせん断流の下で、P-セレクチン糖タンパクリガンド-1(PSGL-1)を発現する白血球はP-セレクチンとの非常に一過性の結合を形成し、炎症を起こした内皮細胞の表面に発現し得る。このプロセスは、白血球が炎症部位に移行するために重要です2.さらに、PSGL-1はまた、P-selectin3との関与時に転がり接着カスケードのその後の堅接着段階を引き起こすことができるメカノイアセンセプターである。
CAM機能に影響を与える遺伝子変異は、白血球接着欠損症(LAD)の稀な疾患など免疫系に深刻な影響を及ぼし、転がりを媒介する接着分子の機能不全が重度の免疫不全個体4,5,6に至る。さらに、循環腫瘍細胞は、同様の圧延過程に従って移行することが示されており、転移7,8に至る。しかし、細胞の転がりは速く動的であるため、従来の実験的メカノバイオロジー法は、細胞転動時の分子相互作用を研究するのには適していません。原子間力顕微鏡や光ツイーツァーなどの単一細胞および単一分子操作法は、P-selectinのPSGL-1とPSGL-1との力依存相互作用などの分子相互作用を単一分子レベル9で研究することができたが、細胞転動中の生きた接着事象の調査には不向きである。さらに、インビトロで特徴付けられる相互作用は、生体内での分子接着に関する質問に直接答えることができない。例えば、細胞が本来の環境で機能している場合、生物学的に関連する分子張力範囲は何ですか?粘着力学simulation10や単純定常モデル11のような計算方法は、特定の分子の詳細を捉え、それらが転がり行動にどのように影響するか、モデリングパラメータおよび仮定の精度に大きく依存している。牽引力顕微鏡のような他の技術は細胞の移動の間に力を検出することができるが、十分な空間分解能または分子張力に関する定量的な情報を提供しない。これらの技術のいずれも、自生環境における細胞機能と挙動に直接関係する分子力の時間ダイナミクス、空間分布、および大きさの不均一性の直接的な実験的観察を提供することはできません。
そのため、転がり接着性の理解を深める上で、選択イン媒介相互作用を正確に測定できる分子力センサを実装することが重要です。ここでは、PSGL-1被覆ビーズが機能性テンションゲージテザー(TG)13 を提示する表面上に転がされる接着フットプリントassay12のプロトコルについて説明する。これらのTGは、蛍光読み出しの形で破裂事象の永久的な歴史をもたらす不可逆的なDNAベースの力センサーです。これは、TGT(dsDNA)の破裂後、破裂したTGT(ssDNA)を蛍光標識された相補鎖で標識することによって達成されます。このシステムの大きな利点の1つは、回折限と超解像イメージングの両方との互換性です。蛍光標識された相補鎖は、回折限定画像撮影のために永久に結合(>12 bp)、またはDNA PAINTを介した超解像イメージングのために一過性(7-9 bp)結合することができる。これは、TGがアクティブローリング中に破裂する際にローリング接着を研究するのに理想的なシステムですが、蛍光読み出しは転がり後に分析されます。また、2つのイメージング方法は、ローリング接着を調査する自由度をユーザに提供します。典型的には、回折限定画像は蛍光強度13を介して分子破裂力を抽出するのに有用であるが、超解像イメージングは受容体密度の定量的分析を可能にする。このアプローチは、圧延接着のこれらの特性を調査する能力を持ち、せん断流下の転動細胞の分子接着に関する力制御機構を理解するための有望なプラットフォームを提供する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-channel drill guide</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>4-holes slide</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>BDH1101-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic spacer</td>
			<td>Custom made</td>
			<td></td>
			<td>Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder.</td>
		</tr>
		<tr>
			<td>Aluminium chip holder</td>
			<td>Custom made</td>
			<td></td>
			<td>Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread.</td>
		</tr>
		<tr>
			<td>Aminosilane</td>
			<td>AlfaAesar</td>
			<td>L14043</td>
			<td>CAS 1760-24-3</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution</td>
			<td>Cytiva HyClone</td>
			<td>SV3007901</td>
			<td>Pen/Strep/Fungiezone</td>
		</tr>
		<tr>
			<td>Beads, ProtG coated polystyrene</td>
			<td>Spherotech</td>
			<td>PGP-60-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>VWR</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer, DNA PAINT</td>
			<td></td>
			<td></td>
			<td>0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Buffer, T50M5</td>
			<td></td>
			<td></td>
			<td>10mM Tris, 50 mM NaCl, 5 mM MgCl2</td>
		</tr>
		<tr>
			<td>Buffer, Rolling</td>
			<td></td>
			<td></td>
			<td>HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA</td>
		</tr>
		<tr>
			<td>Buffer, Wash</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>VWR</td>
			<td>BDH9224</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>VWR</td>
			<td>10062-868</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated sulfuric acid</td>
			<td>VWR</td>
			<td>BDH3072-2.5LG</td>
			<td>95-98%</td>
		</tr>
		<tr>
			<td>Coverslip holding tweezers</td>
			<td>Techni-Tool</td>
			<td>758TW150</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond-coated drill bits</td>
			<td>Abrasive technology</td>
			<td>C5250510</td>
			<td>0.75 mm diamond drill</td>
		</tr>
		<tr>
			<td>DNA, amine-ssDNA (top strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGGAGGG /3AmMO/</td>
		</tr>
		<tr>
			<td>DNA, biotin-ssDNA (bottom strand)</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>/5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG</td>
		</tr>
		<tr>
			<td>DNA, imager strand for DNA-PAINT</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>GAGGGAAA TT/3Cy3Sp/</td>
		</tr>
		<tr>
			<td>DNA, imager strand for permanent labelling</td>
			<td>IDT DNA</td>
			<td>Custom oligo</td>
			<td>CCGGGCGACGCAGG /3Cy3Sp/</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>237</td>
			<td>3/4 inch width, permanent double-sided tape</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermofisher</td>
			<td>15575020</td>
			<td>0.5 M EDTA, pH 8.0</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Gorilla</td>
			<td>42001</td>
			<td>5 minute curing time</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Avantor</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GelGreen</td>
			<td>Biotium</td>
			<td>41005</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>VWR</td>
			<td>BDH3094-2.5LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass, Microscope slide</td>
			<td>VWR</td>
			<td>48300-026</td>
			<td>75 mm x 25 mm x 1 mm</td>
		</tr>
		<tr>
			<td>Glass, Staining jar</td>
			<td>VWR</td>
			<td>74830-150</td>
			<td>Wheaton Staining Jar (900620)</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt solution (HBSS)</td>
			<td>Lonza</td>
			<td>04-315Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber slide support</td>
			<td>Custom made</td>
			<td></td>
			<td>3D printed with ABS filament</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>VWR</td>
			<td>BDH7690-1</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>Inlets/outlets</td>
			<td>Custom made</td>
			<td></td>
			<td>Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm&#160; polyethylene tubing (Tygon, I.D. 1/32&#8221; O.D. 3/32&#8221;) into each hollow setscrew</td>
		</tr>
		<tr>
			<td>Iscove Modified Dulbecco Media (IMDM)</td>
			<td>Lonza</td>
			<td>12-722F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>BDH9244</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Ni-NTA beads</td>
			<td>Invitrogen</td>
			<td>10103D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mailer tubes</td>
			<td>EMS</td>
			<td>EMS71406-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Bio-Spin P-6 Gel Columns</td>
			<td>Biorad</td>
			<td>7326200</td>
			<td>In SSC Buffer</td>
		</tr>
		<tr>
			<td>PEG</td>
			<td>Laysan Bio</td>
			<td>MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-biotin</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>VWR</td>
			<td>470302-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein, Protein G</td>
			<td>Abcam</td>
			<td>ab155724</td>
			<td>N-terminal His-Tag and C-terminal cysteine</td>
		</tr>
		<tr>
			<td>Protein, P-selectin-Fc</td>
			<td>R&#38;D System</td>
			<td>137-PS</td>
			<td>Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF</td>
		</tr>
		<tr>
			<td>Protein, Streptavidin</td>
			<td>Cedarlane</td>
			<td>CL1005-01-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Syringe</td>
			<td>Harvard Apparatus</td>
			<td>704801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ward&#8217;s Science</td>
			<td>470302-444</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>97061-274</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermofisher</td>
			<td>22322</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>81520</td>
			<td>Syringes with PTFE luer lock, 5 mL</td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>BD</td>
			<td>305115</td>
			<td>Precision Glide 26 G, 5/8 Inch Length</td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>VWR</td>
			<td>BDH4502-500GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing, Adaptor</td>
			<td>Tygon</td>
			<td>ABW00001</td>
			<td>Formulation 3350, I.D. 1/32&#8221;; O.D. 3/32&#8221;</td>
		</tr>
		<tr>
			<td>Tubing, Polyethylene</td>
			<td>BD Intramedic</td>
			<td>427406</td>
			<td>Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>93773</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging molecular adhesion in cell rolling by adhesion footprint assay

転動接着は、選択的に媒介される相互作用によって促進され、炎症部位に白血球を採用する上で非常に動的で受動的な運動性である。この現象は、血流が内皮細胞の転動で白血球を押し出す後毛細血管小胞で起こる。安定した転がりは、接着結合形成と機械的に駆動された解離との微妙なバランスを必要とし、細胞が流れの方向に転がり続けながら表面に付着したままにする。比較的静的な環境で発生する他の接着プロセスとは異なり、転がり細胞は毎秒数十ミクロンで数千ミクロンにわたって移動するので、圧着は非常にダイナミックです。その結果、トラクションフォース顕微鏡などの従来のメカノバイオロジー法は、短いタイムスケールと高感度が要求されるため、個々の接着事象および関連する分子力を測定するのには適していません。ここでは、P-セレクチン:分子レベルでの圧延接着におけるPSGL-1相互作用をイメージする接着フットプリントアッセイの最新の実装について説明します。この方法は、不可逆的なDNAベースの張力計テザーを利用して、蛍光トラックの形態で分子接着事象の永久履歴を生成する。これらのトラックは、(1)大きな視野を生み出すために何千もの回折限画像をつなぎ合わせ、長さ数千ミクロンにわたって各転球細胞の接着フットプリントを抽出し、(2)DNA-PAINTを実行して小さな視野内の蛍光トラックの超解像画像を再構築するという2つの方法で画像化することができます。本研究では、接着フットプリントアッセイを用いて、異なるせん断応力で転がるHL-60細胞を研究した。そうすることで、P-セレクチンの空間分布をイメージすることができました:PSGL-1相互作用と蛍光強度を通じて分子力に関する洞察を得ることができました。このように、この方法は、分子レベルでの圧延接着に関与する種々の細胞表面相互作用の定量的調査のための基礎を提供する。

上記のプロトコルは、接着フットプリントアッセイの実験手順を説明する。一般的な実験ワークフローを 図1に示し、流れチャンバアセンブリ(図1A)から表面官能化(図1B)および実験およびイメージングステップ(図1C)までを示す。
図2 は、ProtG-ssDNAバイオコンジ?...

Watch this Scientific Journal Video about Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay at JoVE.com

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

このプロトコルは、高速細胞圧延接着中に接着イベントを画像化するために、接着フットプリントアッセイを実行するための実験的手順を提示する。

接着フットプリントアッセイによる細胞転動における分子接着性のイメージング

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

このプロトコルは、空間的にマッピングし、分子レベルでのローリング接着を定量化する以外に、分子力と細胞転動挙動の間の接続を確立するのに役立ちます。この技術は、実際の細胞転動中の個々の接着事象を研究し、生理学的関連分子接着力を直接測定することを可能にする。各工程で適切な濃度とインキュベーション時間を使用した表面調製は、高品質の表面機能化を実現するために重要です。バイオコンジュゲーションの品質と適切な条件も重要です。視覚的なデモンストレーションは、研究者がこのプロトコルを複製するのを助けるために重要です。特に、フローチャンバアセンブリや後処理画像などのステップは、視覚的なデモンストレーションから大きなメリットを得られます。カミソリの刃で両面テープの両側に少量のエポキシを薄く広めることから始めます。レーザーを使用して、エポキシコーティングされたテープをカットして4つのチャンネルを作成します。4ホールスライドとPEGカバースリップの間にエポキシテープを挟んでフローチップを作成します。ピペットチップを使用して、チャネルの長さに沿って穏やかな圧力をかけて良好なシールを作成し、エポキシを最低1時間硬化させます。各チャンネルの開口部がアダプタの中心に配置されるようにチップを位置合わせします。その後、チップの上に2つの透明なアクリルスペーサーを置きます。ブロックの中央にしっかりした圧力をかけ、各スペーサーの端にねじを付けます。入口をブラケットの反対側のネジ穴にねじ込み、透明なアクリルブロックを通してシール状態を監視します。200マイクロリットルの洗浄バッファーをチャンバに流し、漏れを確認します。気泡がチャネルに形成される場合は、積極的に気泡を除去するために、洗浄バッファーの追加の200マイクロリットルを押します。非特異的結合を防止し、湿度室で10分間インキュベートするために、40マイクロリットルのBSAをフローチャンバーに加えます。インキュベーション後、フローチャンバーにTween 20で40マイクロリットルを加え、再び10分間インキュベートして非特異的結合をさらに低減する。次に、200マイクロリットルの洗浄バッファーでチャネルを洗浄し、すべてのパッシベーション剤を除去します。チャンバー表面機能化のために、ストレプトアビジンを40マイクロリットルの流れチャンバに加え、20分間インキュベートしてから、200マイクロリットルの洗浄バッファーでチャンバーを洗浄します。次に、40マイクロリットルのハイブリダイズプロテインG-TGTをフローチャンバーに加え、20分間インキュベートします。洗浄バッファーで洗浄した後、40マイクロリットルのプロテインG-TGTトップストランドを加え、20分間インキュベートして、表面上の任意の未ハイブリッド化TGT底鎖を完了し、洗浄バッファーで洗浄します。最後に、40マイクロリットルのP-セレクチン-Fcをフローチャンバーに加え、洗浄バッファーで洗浄する前に60分間インキュベートします。5ミリリットルのガラス注射器にローリングバッファーを充填し、シリンジの側面をタップして取り外し、先端に向かって浮かぶように気泡を押し出します。ポリエチレンチューブの200ミリメートル部分に滅菌針を挿入した後、ガラス注射器に針を接続します。シリンジをシリンジポンプに固定し、気泡がチャネルに入るのを防ぐためにプランジャー側が上昇するようにシリンジポンプを傾けます。チューブの端部を流れチャンバの入口に挿入します。ポリエチレンチューブの別の200ミリメートル片の一方の端を出口に挿入し、もう一方の端を廃棄物ビーカーに浸します。細胞懸濁液サンプルと遠心分離機の1~2ミリリットルを細胞にペレットに取る。培地を取り出し、500マイクロリットルのローリングバッファーで細胞を静かに再懸濁します。慎重に流入口とフローチャンバーに細胞懸濁液の出口とピペット40マイクロリットルからチューブを切断します。前述のとおりにチューブを再接続し、フロー チャネルに気泡が入らないようにします。所望の流速でシリンジポンプを開始して細胞の転がり実験を開始します。10Xの目的を持つ暗視野顕微鏡を使用して細胞の転がりを観察する。実験が完了したら、表面が細胞フリーになるまで1時間あたり100ミリリットルでローリングバッファーを注入することによってチャネルから細胞を取り除きます。DNA-PAINTによるローカルトラックのイメージング用に、DNA-PAINTバッファで作製した40マイクロリットルのDNA-PAINTイメージャー鎖をチャネルに加えます。本文原稿に記載された条件を用いて、全内部反射蛍光顕微鏡を行う。スーパー解像度イメージをローカライズしてレンダリングします。永久的なラベル付けによって長いトラックを撮像するために、永久的なイメージャーの鎖を加え、T50M5の緩衝装置の120秒のインキュベート。次に、200マイクロリットルの洗浄バッファーを注入してチャネルを洗浄します。励起レーザーをオフにして画像を記録し、カメラの背景ノイズを取得します。TIRF顕微鏡法によるグリッドパターンの大きな領域を画像化します。400 x 50画像の領域をスキャンし、ImageJプログラムを使用して最大10,000枚の画像を含む個々のTIPファイルに生データを分割するように顕微鏡をプログラムします。照明プロファイルを使用してすべての画像を平坦化し、画像をステッチするMISTプラグインを使用します。タンパク質G ssDNAバイオコンジュゲーション特性の結果は、タンパク質GのssDNAおよびイミダゾール溶出緩衝液スペクトルが生体共役生成物スペクトルに適合するためのssDNAに対するタンパク質Gの1つの比率にほぼ1つであることを示した。ネイティブPAGEは、単量体タンパク質Gバンドと一致する明るい緑色のバンドを示すバイオコンジュゲーションを確認するために使用され、良好な収率を示した。シングルモードファイバから導入されたTIRF照明プロファイルは、一般的に視野の中央で明るく、エッジの周りに暗くなります。不均一な照明を補正し、定量的な分析のために画像を平らにするために、照明プロファイルは、個々のフレームの数千を平均することによって決定しました。平坦化された画像は、生プロファイルと照明プロファイルの両方からカメラノイズを引き出し、照明プロファイルによって正規化することによって生成されました。生のステッチは、未修正の画像に対応する明確な周期パターンを示し、平坦化された画像からステッチされた同じ視野は平らな背景を生み出した。ランプアップフロープロファイルを使用して、典型的な単細胞接着フットプリントを見ることができる細胞転がりおよび収量蛍光トラックをもたらすせん断応力の範囲を決定しました。コントラストが不十分な蛍光トラックの最適で最適な結果、過度のトラック密度、最適なトラック密度とコントラスト、回折限定およびDNA-PAINTイメージングが示されています。60分以上のインキュベーション時間は、表面の機能化を確保し、適切なチャンバー構造は、機能化された表面を損傷する気泡の通過を防止します。この手順は、圧延接着に関与する分子力の定量的分析に適用され、研究者は異なる細胞タイプの圧延挙動を理解することができます。この技術により、研究者は、単一分子およびメカノ生物学研究のさらなる進歩につながる高速接着事象に関する新たな疑問を探求することができます。

imaging-molecular-adhesion-in-cell-rolling-by-adhesion-footprint-assay

Research

JoVE Journal

Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

生物学

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

アフリカツメガエルの胚および細胞接着アッセイのアセンブリから主催し、動物極の外植片の解剖

Title Cell Encapsulation by Droplets

滴タイトルセルのカプセル化

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

マイクロ流体フローセルを使用して流れの下で血小板粘着と凝集

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

カルシウムの役割を評価するためのイメージングアッセイを生きる<sup&gt; 2 +</sup孔形成毒素の傷の修復&gt;とスフィンゴミエリナーゼ

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

マウスで有酸素運動によってオートファジーのアクティブ化

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

磁気ビーズを使用した機械的張力に応答して細胞表面接着リフォームの分析

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...