Name: a method of targeted cell isolation via glass surface functionalization
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

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.

1. Rengjøring glassoverflaten og Forbereder reagenser <ol><li> Plasser en glassoverflate i en oksygen plasma maskin i 5 min ved 50% effekt for å rense det. </li><li> Fremstille 2,5 ml 2% rekonstituert (3-aminopropyl) triethoxysilane (APTES) løsning ved å tilsette 50 ul APTES og 2,45 ml etanol i et konisk rør. </li></ol> 2. APTES og DSB funksjon <ol><li> Legg APTES løsning til overflatene. Pipetter 150 ul per brønn til 8-brønners plater. Pipetter 100 ul per brønn i 24 brønners plater...

<ol>
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Forbedringer i celleisolasjonsteknikker fremmer vitenskapelige studier i struktur-funksjon relasjoner i nevrovitenskap 18, stamcelle programmering i regenerativ biologi, og angiogenic signalering i vaskulær biologi 19. Faktisk, primære cellekultur 20 (f.eks HUVECs) i vaskulær biologi skjer primært ved bruk av celleisoleringsteknikker. Cell isolasjon ble også nylig brukt for kvantitativ flow (qFlow) cytometri analyse av plasmamembranreseptorer 3,14,15,19,21. Imidl...

Nåværende benk-top Celleseparasjonsanordningene tilnærminger (f.eks fluorescens aktivert celle sortering 1, laser capture mikro-disseksjon 2 analysatorer, magnetiske kuler separasjon 1) kan ta flere timer med forberedelser og sortering. Disse store tidsskalaer kan påvirke fysiologiske respons og uttrykk nivåer, noe som resulterer i analyser som ikke er representative for den fysiologiske responsen tre. Systemer er nødvendig som raskt og effektivt kan isolere spesifikke celletyper uten å avbryte celleoverflate reseptor-nivåer for å forbedre celleisolering og berikelse for biomedisinske anvendelser. Derfor er begrunnelsen for vår tilnærming er å utvikle en mild metode for celleisolasjon. Den &quot;lab on a chip&quot; -konseptet gir løfte om størrelsesordener raskere (timer til minutter) celle isolasjon, og oftest innebærer å fange celler på en overflate og slippe celler eller intracellulær contents gjennom fysisk 4,5 eller kjemiske metoder 6. Selv om disse metodene har noen fordeler som for eksempel å identifisere protein 7,8 uttrykk, å identifisere RNA-ekspresjon 9-11, eller til og med tilveiebringe celler for in vitro kultur 12,13, mange av disse teknikker kan ikke oversettes til diagnostikk, slik som cellereseptoren profilering på grunn til sine ikke-fysiologiske miljøer. Enzymatiske løfte agenter som kollagenaser kan også påvirke disse reseptor mengder 14,15, noe som betyr celle reseptor kvantifisering teknikker som bruker disse løfte agentene vil ikke generere nøyaktige fysiologiske data. Cellulær lyse hindrer differensiering mellom de innfødte overflatereseptorer, og de ​​som tidligere ble internalisert 16. Denne protokollen beskriver en rask og skånsom tilnærming for celleisolasjon.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.</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 
			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 
			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 
			(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 5mg 
			[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 100mL at room temperature</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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>10% fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>03-600-511</td>
			<td>Stored in 500mL at &#60; -10&#8304;C</td>
		</tr>
		<tr>
			<td>1% Penicillin&#8211;Streptomycin</td>
			<td>Life Sciences Storeroom at UIUC</td>
			<td>17602e</td>
			<td>Supplier: VWR 
			Stored in 100 ml at 4&#8304;C</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>12-565-58</td>
			<td>Small 23cm 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 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 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.

Ved hjelp av denne protokollen vi vise celle fangst (figur 3A) og celle frigjøring (Figur 3C) av MCF7GFP celler samt levende cellekontroller (figur 4). Vi kvantifisert cellen fange som 60% og 80% ble gitt ut (Figur 3C). Når vi utvidet denne tilnærmingen til en blanding av RAW 264,7 makrofager og MCF7GFP-celler, ble 50% av RAW makrofager fanges opp (fig. 3D) og 80% av RAW makrofager var frigivelse med ...

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.

En metode for målrettet Cell Isolation via Glass Surface funksjon

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

Research

JoVE Journal

Cancer Research

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

En Mimic av svulstens mikromiljø: En enkel metode for å generere beriket celle populasjoner og Gransker inter Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Anticancer Effekten av fotodynamisk behandling med Lung Cancer-Målrettet Nanopartikler

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Isolering av sirkulerende tumorceller i en ortotopisk musemodell av kolorektal kreft

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Generasjon og isolering av cellen syklus-arrestert celler med kompliserte Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Overflaten forbedret resonans Raman spredning Nanoprobe Ratiometry for å oppdage mikroskopiske eggstokkreft via folat reseptor målretting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Paramyxoviruses for svulst målrettede Immunomodulation: Design og evaluering Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Isolering av Stem-lignende celler fra 3-dimensjonale spheroid kulturer

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Isolering og funksjonell vurdering av humane brystkreftstamceller fra celle- og vevsprøver

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Cellulær profilering med høy gjennomstrømning av målrettede proteinnedbrytningsforbindelser ved bruk av HiBiT CRISPR-cellelinjer

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

En Murine Ommaya Xenograft-modell for å studere direkte målrettet terapi av leptomeningeal sykdom

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...