The authors thank M. Gordon of the Queen's University Biomedical Imaging Centre for assistance. This work was supported by operating grants from the Cancer Research Society of Canada (19439) and the Canadian Institutes for Health Research (MOP-142303) (LMM), and by Ontario Graduate Scholarships and studentships from the Terry Fox Research Institute Training Program in Transdisciplinary Cancer Research (SMM, EYL), and by a Craig Jury Summer Studentship (SMM).

<ol>
	<li>Zimmermann, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+vs.+three-dimensional+in+vitro+tumor+migration+and+invasion+assays.">Two-dimensional vs. three-dimensional in vitro tumor migration and invasion assays.</a> Methods Mol Biol. 986 (986), 227-252 (2013).</li><li>Albini, A., Noonan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'chemoinvasion'+assay,+25+years+and+still+going+strong:+the+use+of+reconstituted+basement+membranes+to+study+cell+invasion+and+angiogenesis.">The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis.</a> Curr Opin Cell Biol. 22 (5), 677-689 (2010).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat. Rev. Mol. Cell Biol. 8 (10), 839-845 (2007).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends Biotechnol. 31 (2), 108-115 (2013).</li><li>Carletti, E., Motta, A., Migliaresi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> Methods Mol Biol. 695, 17-39 (2011).</li><li>Foty, R. <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+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J Vis Exp. (51), (2011).</li><li>Handschel, J. G., 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=Prospects+of+micromass+culture+technology+in+tissue+engineering.">Prospects of micromass culture technology in tissue engineering.</a> Head Face Med. 3, 4 (2007).</li><li>Stuckhoff, A. P., DelValle, L., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuronal Cell Culture Methods and Protocols Vol. 1078 Methods in Molecular Biology. 1078, 119-132 (2013).</li><li>Stewart, G. J., Wang, Y., Niewiarowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+protects+the+ability+of+anchorage-dependent+cells+to+adhere+following+isolation+and+holding+in+suspension.">Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.</a> Biotechniques. 19 (4), 598-604 (1995).</li><li>Vinci, M., 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=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biol. 10, 29 (2012).</li><li>Nagelkerke, A., Bussink, J., Sweep, F. C. G. J., Span, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+of+breast+cancer+cells:+How+to+go+three-dimensional.">Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional.</a> Anal. Biochem. 437 (1), 17-19 (2013).</li></ol>

The authors have nothing to disclose.

私たちは、安価で広く入手可能な試薬を用いたin vivo組織のアーキテクチャをモデル化するために、3Dスフェロイドを生産するための迅速かつ柔軟な方法を提示します。我々のプロトコルは、細胞凝集を媒介し、細胞単層の形成を最小限にするためにMC 8,9の非細胞毒性および接着促進特性を利用します。動物源から単離されたタンパク質ベー?...

腫瘍細胞の挙動の生物学的に関連する評価は、これらが十分に生体内で見つかった細胞微小環境を反映していないため、部分的には、従来の2次元（2D）細胞培養法を用いて挑戦されています。培養物（ 例えば 、Boydenチャンバーアッセイ）に細胞外マトリックス成分を組み込んだ別のアプローチは、生体内の組織環境の複数の生理的に代表的なものです。しかしながら、それらは個々の細胞の挙動の評価に限定することができ、組織または腫瘍増殖1、2、3に寄与する細胞-マトリックス及び細胞-細胞間相互作用のインビボでの組み合わせで複合体を再現しません。 多細胞スフェロイドの使用は、より正確にin vivoでの細胞増殖1のコンパクトなアーキテクチャを再現最近のアプローチであります4。スフェロイドは、正常細胞の細胞-マトリックス相互作用を調査するために使用することができるだけでなく、転移性増殖または薬剤耐性4のように、腫瘍進行の特性をモデル化するために、腫瘍の類似体として作用することができます。 スフェロイドは、単一細胞クラスター（ 例えば 、懸滴、遠心分離法）6,7を形成するために、複数の細胞の凝集を促進することにより、より迅速マトリックス5内に埋め込まれた単一細胞の増殖により形成された、又はされてもよいです。既存の細胞凝集技術は、高価な材料や特殊な装置を必要とし得ます。加えて、これらのスフェロイドは、サイズ及び形態の広い範囲を有し、成長条件または困難な処置間の比較を行う、大量に製造することが困難であってもよいです。最後に、これらの方法によって生成されたスフェロイドは、タンパク質extracelから単離することは困難ですそれらは、他のアプリケーションで使用するために埋め込まれたlularマトリックス。 ここでは、市販のU底細胞撥プレートと不活性接着促進マトリックス、メチルセルロースを使用して、一貫サイズのスフェロイドの急速な形成のための堅牢かつ容易に修正細胞凝集方法を説明します。一旦形成されると、これらの多細胞スフェロイドを容易に広範囲の用途での使用のために単離されます。プロトコルはまた、容易に他の細胞プロセスを評価するために使用され得る細胞増殖を介してスフェロイドを生成するように適合されています。ここでは、これらの二つの異なる回転楕円体形成プロトコルのアプリケーション例として、免疫蛍光染色によって定量し、細胞浸潤アッセイ、およびアノイキスアッセイを示します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>21114</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of calcium chloride</td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Used for PBS* wash buffer; Do not autoclave PBS* wash buffer upon addition of magnesium chloride</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Spheroid Formation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well U-bottom Cell-Repellent Plate</td>
			<td>Greiner Bio-One</td>
			<td>650970</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td>For culturing SH-SY5Y, PANC-1, TPC-1 cell lines</td>
		</tr>
		<tr>
			<td>F12K Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>2112722</td>
			<td>For culturing TT cell line</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F1051</td>
			<td>Filter prior to use to remove particulate contaminants</td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M7027</td>
			<td>Prepare in water to 100 mg/mL</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium</td>
			<td>Sigma-Aldrich</td>
			<td>R8758</td>
			<td>For culturing HCT-116, BxPC-3 cell lines</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605028</td>
			<td>Dissociation buffer</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Invasion Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Type I Collagen</td>
			<td>Corning Incorporated</td>
			<td>354231</td>
			<td>Stock 3.1mg/ml; Maintain on ice when in use</td>
		</tr>
		<tr>
			<td>DMEM Phenol Red Free Low Glucose&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054-20</td>
			<td>Less background fluorescence compared to Phenol Red supplemented medium</td>
		</tr>
		<tr>
			<td>Glial Cell Line Derived Neurotrophic Factor</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>Chemoattractant</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For Immunofluorescence Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#1.5 Coverglass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72225-01</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>Alexa-Fluor 488 Phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>Used to stain actin at 1:200</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bioshop Canada Incorporated</td>
			<td>ALB001</td>
			<td>Used in BSA blocking buffer</td>
		</tr>
		<tr>
			<td>Dabco 33-LV</td>
			<td>Sigma-Aldrich</td>
			<td>290734</td>
			<td>Antifade</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Bioshop Canada Incorporated</td>
			<td>GLY001</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>Freeware, NIH</td>
			<td>-</td>
			<td>Used for image analysis&#160;</td>
		</tr>
		<tr>
			<td>Microslides</td>
			<td>VWR International</td>
			<td>48312-024</td>
			<td>For mounting excised spheroids</td>
		</tr>
		<tr>
			<td>MOWIOL 4-88</td>
			<td>EMD-Millipore</td>
			<td>475904</td>
			<td>Used in MOWIOL mounting medium</td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>EMD-Millipore</td>
			<td>PX0055-3</td>
			<td>Used in fixation buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioshop Canada Incorporated</td>
			<td>TRX777</td>
			<td>Used in permeabilization buffer</td>
		</tr>
	</tbody>
</table>

an efficient and flexible cell aggregation method for 3d spheroid production

単層細胞培養物は十分に複雑な細胞-細胞及び細胞-マトリックス相互作用に関与する組織のインビボ挙動をモデル化しません。三次元（3D）細胞培養技術は、接着細胞培養物の欠点に対処するために開発され、最近の技術革新です。 インビトロ組織類似体を生成するためのいくつかの技術が開発されてきたが、これらの方法を確立すること、高価、しばしば複雑であり、特殊な装置を必要とし、一般的にのみ、特定の細胞型との互換性により制限されます。ここでは、腫瘍の様々な成長と正常細胞株と互換性のある一貫したサイズの多3Dスフェロイドに細胞を集約するための迅速かつ柔軟なプロトコルを記述します。我々は、足場非依存性スフェロイドの生成を促進し、高度に再現可能な方法で細胞単層の形成を防止するために、血清およびメチルセルロース（MC）の種々の濃度を使用します。 INDIVのための最適条件idual細胞株はスフェロイド形成培地中MCまたは血清濃度を調整することによって達成することができます。生成された3Dスフェロイドは、細胞シグナル伝達または遺伝子発現研究、候補薬物のスクリーニング、またはそのような腫瘍細胞の浸潤および遊走などの細胞プロセスの研究に含めた幅広い用途での使用のために収集することができます。プロトコルはまた、容易に単一細胞からクローンスフェロイドを生成するように適合され、足場非依存性増殖およびアノイキス耐性を評価するために適合させることができます。全体として、我々のプロトコルは、組織の三次元微小環境を再現し、正常細胞および腫瘍細胞のインビボ増殖をモデル化するために、3D細胞スフェロイドを生成し、利用するための簡単に修正する方法を提供します。

我々は、細胞撥プレートとMCを補充したスフェロイド形成媒体を使用して個別のスフェロイドを生成するための柔軟で効率的な方法を説明します。 MCおよび血清の適切な条件の下では、個々の細胞が落ち着くとウェル底への最小限の遵守とスフェロイドを形成するために、ウェルの中央で一緒に接着します。このプロトコルを使用して、スフェロイドは、細胞株の様?...

Watch this Scientific Journal Video about An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production at JoVE.com

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

3Dスフェロイド生産のための効率的で柔軟な細胞凝集法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

an-efficient-flexible-cell-aggregation-method-for-3d-spheroid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

23.6K ビュー.  Queen's University Cancer Research Institute. Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions. 

ビデオ: An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

生物学

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

統合欠陥レンチウイルスベクターにパッケージ化された転写調節睡眠美容システムによって効率的な体外転位法

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共焦点顕微鏡を明らかに細胞表面受容体画像相関分光法による凝集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

3Dスフェロイドモデルにおける時間分解蛍光イメージングとがん細胞浸潤解析

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

シロイヌナズナなどのうどんこ病感染表現型を正確に評価するための簡便かつ柔軟な接種法

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

蛍光相関分光法を用いたタンパク質凝集の検出

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

シロイヌナズナ種子の表面滅菌のための高スループット、堅牢で高い時間融性の高い方法

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

ハイコンテンツスクリーニングおよび分析アプリケーションのためのスフェロイドの大規模生産のための堅牢な方法

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

化学発光アッセイによるイネの免疫応答における活性酸素種産生のリアルタイム検出

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...