We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.

<ol>
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The authors have nothing to disclose.

細胞分離技術の改善は、血管生物学19で細胞の再生生物学でのプログラミング、および血管新生シグナル伝達幹、神経科学18における構造と機能の関係に科学的研究をfurthers。実際、初代細胞培養物20（ 例えば 、HUVECを）血管生物学のは、主として細胞分離技術を使用して行われます。細胞の単離は、最近細胞膜受容体3,14,15,19,21の定量的な流れ（qFlow）?...

現在のベンチトップ細胞分離手法（ 例えば 、蛍光活性化細胞は1ソート、レーザーキャプチャーマイクロダイセクション2、免疫磁気ビーズ分離1）が準備し、ソートの数時間かかることがあります。これらの大きな時間スケールは、生理学的応答3の代表ではない分析の結果、生理学的応答および発現レベルに影響を与えることができます。システムは、迅速かつ効率的な生物医学的用途のための細胞の単離及び濃縮を改善するために、細胞表面受容体レベルを中断することなく、特定の細胞型を分離することができる必要とされています。したがって、我々のアプローチの理論的根拠は、細胞単離のための穏やかなアプローチを開発することです。 「ラボオンチップ」概念が速く（時間・ツー・分）細胞単離を桁違いの約束を提供し、最も頻繁に表面上に細胞を捕捉し、細胞や細胞内のコンテを解放することを含みます4,5物理的または化学的方法6を通じてNTS。これらのアプローチは、このようなRNA発現9-11、あるいはin vitro培養12,13のための細胞を提供する識別、タンパク質7,8式を識別するなど、いくつかの利点を提供していますが、これらの技術の多くは、このような原因で細胞受容体プロファイリングなどの診断に翻 ​​訳することはできませんそれらの非生理的な環境です。このようなコラゲナーゼなどの酵素リフティング剤もこれらのリフティングエージェントが正確な生理学的データを生成しません使用する細胞受容体の定量化技術を意味し、これらの受容体の量14,15に影響を与えることができます。細胞溶解は、天然の表面受容体との間の差別化を防止し、あらかじめ16を内在したもの。このプロトコルは、細胞単離のための高速かつ穏やかなアプローチを説明しています。

<table><tbody><tr><td>(3-Aminopropyl) triethoxysilane (APTES)</td><td>Acros Organics</td><td>919-30-2</td><td>Used to make 2% APTES solution</td></tr><tr><td>Plasma Cleaner Pico</td><td>Diener</td><td>Model 1</td><td>Cleans surfaces and allows for bonding of PDMS to glass</td></tr><tr><td>d-Desthiobiotin (DSB)</td><td>Sigma</td><td>D20655</td><td>Used as the releasing mechanism in the cellular capture surface.&amp;nbsp;</td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>British Drug Houses (BDH)</td><td>BDH1115-1LP</td><td>Dissolves the DSB into solution</td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td><td>Thermo-Scientific</td><td>5g: 22980&lt;br/&gt; 25g: 22981</td><td>Activates carboxylic acids and allows binding of proteins to glass surface.</td></tr><tr><td>uncoated 8-well culture slide</td><td>BD Falcon</td><td>Case of 24: 354118&lt;br/&gt; Case of 96: 354108</td><td>Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments</td></tr><tr><td>Glass bottom 24-well plates</td><td>MatTek</td><td>P24G-0-13-F</td><td>Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments</td></tr><tr><td>Mercaptoethanol</td><td>Science Lab</td><td>60-24-2</td><td>Used to quench reaction between EDC and DSB</td></tr><tr><td>4-Morpholinoethanesulfonic acid hydrate&lt;br/&gt; (MES Hydrate 99%)</td><td>Fisher Scientific</td><td>AC172590250</td><td>Used to make 0.1 M MES Buffer for use in EDC reaction</td></tr><tr><td>Precision Oven</td><td>Thermo Scientific</td><td>11-475-153</td><td>Used in curing of PDMS and APTES layer.</td></tr><tr><td>Titramax 1000 Shaker</td><td>Heidolph</td><td>13-889-420</td><td>Used to ensure even distribution of APTES on surfaces.</td></tr><tr><td>1x Streptavidin 5 mg [e7105-5mg]</td><td>Proteo Chem</td><td>9013-20-1</td><td>Biotin-binding protein. May cause irritation.</td></tr><tr><td>5 cm Glass Dish</td><td>Fisher Scientific</td><td>08748A</td><td>Used in HUVEC studies as well as future profiling studies.</td></tr><tr><td>14 cm Petri Dish with Cover</td><td>Sigma-Aldrich</td><td>Z717231</td><td>Used to hold samples being functionalized and transport them.</td></tr><tr><td>MCF7-GFP cells</td><td>Cell Biolabs</td><td>AKR211</td><td>Stored in liquid nitrogen</td></tr><tr><td>RAW264.7 mouse macrophages</td><td>ATCC</td><td>TIB-71</td><td>Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen.</td></tr><tr><td>TrypLE</td><td>Life Technologies</td><td>12605036</td><td>Stored in 100 ml at room temperature</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Cell Media Facility at School of Chemical Sciences at UIUC</td><td>50003PC</td><td>Supplier: Corning</td></tr><tr><td>Nonessential amino acids</td><td>Cell Media Facility at School of Chemical Sciences at UIUC</td><td>25-025-CI</td><td>Already added into DMEM by facility. Supplier: Corning.</td></tr><tr><td>Cell scraper</td><td>Fisher Scientific</td><td>12-565-58</td><td>Small 23 cm 50 pack</td></tr><tr><td>Cell Dissociation Solution</td><td>Corning</td><td>MT-25-056CI</td><td>Used to lift cells non-enzymatically for the use in cell experiments</td></tr><tr><td>Hemacytometer</td><td>Hausser</td><td>02-671-54</td><td>Used to count cells for quantification of cell solutions and capture and release effectivity.</td></tr><tr><td>Biotin</td><td>Amresco</td><td>58-85-5</td><td>Used to release cells from surface.</td></tr><tr><td>HBSS</td><td>Created from Recipe</td><td>N/A</td><td>Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. Adapted from Cold Spring Harbor Protocols: In 500&amp;nbsp;ml, use 4&amp;nbsp;g NaCl, 0.2&amp;nbsp;g KCl, 0.0402&amp;nbsp;g Na&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O, 0.03&amp;nbsp;g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; and 0.5&amp;nbsp;g glucose. Add DI water to get to&amp;nbsp;500 ml, filter, and then refrigerate.</td></tr><tr><td>HLA-ABC Antibody</td><td>BioLegend</td><td>311402</td><td>Antibody used to capture MCF7gfp cells</td></tr><tr><td>hIgG Antibody</td><td>BioLegend</td><td>HP6017</td><td>Antibody used to capture MCF7gfp cells</td></tr><tr><td>MCF7 GFP cells</td><td>Cell Biolabs</td><td>AKR-211</td><td>Luminal Breast Cancer line that has been transfected with green fluorescent protein.</td></tr><tr><td>Assorted Conicals</td><td>Thermo-Scientific</td><td>15mL: 12-565-268</td><td>50/15 ml plastic conicals for storing solutions and aliquots.</td></tr><tr><td>Mini-Tube Rotators (End over End Mixer)</td><td>Fisher Scientific</td><td>05-450-127</td><td>Used to incubate antibody and mix other cellular solutions in order to mix</td></tr><tr><td>Axiovert 200M (Fluorescence Microscope)</td><td>Zeiss</td><td>N/A</td><td>Zeiss Axiovert 200 M inverted florescence microscope.</td></tr><tr><td>Zeba Desalting columns</td><td>Thermo-Scientific</td><td>PI-87770</td><td>Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf</td></tr><tr><td>EZ Link Sulfo NHS Low Weight Biotinylation Kit</td><td>Thermo- Scientific</td><td></td><td>Used to biotinylate antibodies to allow them to integrate with the capture surface</td></tr><tr><td>Plate Reader</td><td>BioTek</td><td>Synergy HTX Multimode Reader</td><td>Used to quantitatively measure fluorescent intensity in the titration experiments.</td></tr></tbody></table>

a method of targeted cell isolation via glass surface functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

このプロトコルを使用して、我々は、細胞捕捉（ 図3A）とMCF7GFP細胞ならびに生細胞対照（ 図4）の細胞の放出（ 図3C）を示しています。 60％と80％が放出されたとして、我々は（ 図3C）細胞捕獲を定量化しました。私たちは、RAW 264.7マクロファージおよびMCF7GFP細胞の混合物にこのアプローチを拡張すると、RAWマクロ?...

Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

A Method of Targeted Cell Isolation via Glass Surface Functionalization

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

ガラスの表面機能を介して、標的細胞の単離方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

a-method-targeted-cell-isolation-via-glass-surface

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.3K ビュー.  University of Illinois at Urbana-Champaign. This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process. 

ビデオ: A Method of Targeted Cell Isolation via Glass Surface Functionalization

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

単一のセル下流解析のターゲット細胞前濃縮と全体のゲノム増幅

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

がん研究

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

蛍光顕微鏡を用いたイメージング細胞遊走用接着剤及び可溶性グラデーションを作成する

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

生物工学

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

新規の表面マーカーの組み合わせで識別離および肝前駆サブセットの濃縮

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

発生生物学

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

トラクション力顕微鏡用の細胞培養表面近くのローカライズレポーター蛍光ビーズのための新しい方法

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定化生体分子の単一分子蛍光測定のための自動化システム

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

単離と<em&gt;エクスビボ</emVδ1の&gt;文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT細胞、胸腺外αβT細胞前駆

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

免疫・感染学

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

マクロファージの融合を研究するための光学的品質ガラス表面を加工

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

細菌スクリーニング、選択、および分離のための光分解性ヒドロゲルインターフェース

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

汎用性の高い循環腫瘍細胞を単離するための臨床用マイクロ流体チッププラットフォーム

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Source: Chen S. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of EpCAM-expressing epithelial cancer cells from peripheral blood samples. The technique involves the use of a medical-grade wire functionalized with EpCAM&#160;antibodies to capture the target cells. The captured cells are then subsequently extracted into a buffer solution.

官能基化されたワイヤーベースの標的細胞単離:スパイク末梢血から癌細胞を単離するためのex vivo技術

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
In this ...