Name: induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells
Uploaded: 2020-12-11T13:00:00
Description: Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Forfatterne vil gerne takke Gregg Sealy og Alain Conti for fremragende teknisk bistand og Prof. Zsuzsanna Izsvák fra Max-Delbrück Center i Berlin for venligt at give pSB100X og pT2-CAGGS-Venus plasmid. Dette arbejde blev støttet af Swiss National Sciences Foundation og Europa-Kommissionen inden for rammerne af det syvende rammeprogram. Z.I blev finansieret af Det Europæiske Forskningsråd, ERC Advanced [ERC-2011-ADG 294742].

Procedurerne for indsamling og brug af menneskelige øjne blev godkendt af Den Kantonale Etiske Forskningskommission (nr. 2016-01726).
1. Celleisolation og kulturforhold
<ol><li>Human ARPE-19 cellelinje<ol>
<li>Kultur 5 x 105 ARPE-19 celler, en menneskelig RPE-cellelinje i Dulbeccos Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/Ham's F-12) suppleret med 10% fosterkvægserum (FBS), 80 U/mL penicillin, 80 μg/mL streptomycin og 2,5 μg/mL amphotericin B (komplet medium) ve...

<ol>
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W., Van Houten, B., Conklin, C. A., Jin, G. F., Godley, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+causes+significant+mitochondrial+DNA+damage+in+human+RPE+cells.">Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells.</a> Experimental Eye Research. 68 (6), 765-772 (1999).</li><li>Ma, S., 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=Transgenic+overexpression+of+uncoupling+protein+2+attenuates+salt-induced+vascular+dysfunction+by+inhibition+of+oxidative+stress.">Transgenic overexpression of uncoupling protein 2 attenuates salt-induced vascular dysfunction by inhibition of oxidative stress.</a> American Journal of Hypertension. 27 (3), 345-354 (2014).</li><li>Johnen, S., 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=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 53 (8), 4787-4796 (2012).</li><li>M&#225;t&#233;s, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nature Genetics. 41 (6), 753-761 (2009).</li><li>. Marienfeld Technical information Neubauer-improved Available from: <a target="_blank" href="https://www.marienfeld-superior.com/information-about-our-counting-chambers.html">https://www.marienfeld-superior.com/information-about-our-counting-chambers.html</a> (2020)</li><li>. Electron Microscopy Sciences. Neubauer Haemocytometry Available from: <a target="_blank" href="https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx">https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx</a> (2020)</li><li>Livak, K. J., Schmittgen, T. 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Protokollen præsenteres her tilbyder en tilgang til at analysere anti-oxidative og beskyttende funktion af PEDF og GM-CSF produceret af transfected celler, som kan anvendes til celler transfected med enhver putative gavnlige gen. I genterapistrategier, der har til formål at levere proteiner til væv ved at transplantere genetisk modificerede celler, er det afgørende at indhente oplysninger om niveauet af proteinudtryk, udtryks levetiden og effektiviteten af det udtrykte protein i en model af sygdommen. I vores laborat...

Modellen beskrevet her, tilbyder en nyttig tilgang til at evaluere effektiviteten afbiofarmaceutiske midler til at reducere oxidativ stress i celler. Vi har brugt modellen til at undersøge de beskyttende virkninger af PEDF og GM-CSF på H2O2-medieret oxidativ stress på retinale pigment epitelceller, som udsættes for høje niveauer af O2, og synligt lys, og fagocytose af fotoreceptor ydre segment membraner, genererer betydelige niveauer af reaktive iltarter (ROS)1, 2. De betragtes som en væsentlig bidragyder til patogenesen af avaskulær aldersrelateret makuladegeneration (aAMD)3,4,5,6,7,8. Desuden er der et fald i RPE-syntetiserede neuroprotektive faktorer, specielt pigment epitel-afledt faktor (PEDF), insulin-lignende vækstfaktorer (IGFs), og granulocyte makrofag-koloni-stimulerende faktor (GM-CSF), der fører til dysfunktion og tab af RPE celler, efterfulgt af fotoreceptor og retinal ganglion celle (RGC) død3,4,5 . AMD er en kompleks sygdom, der skyldes samspillet mellem metaboliske, funktionelle, genetiske og miljømæssige faktorer4. Manglen på behandlinger for aAMD er den vigtigste årsag til blindhed hos patienter over 60 år i de industrialiserede lande9,10. Rekonstitutionen af det neuroprotektive og neurogene retinale miljø ved subretinal transplantation af genetisk modificerede RPE-celler, der overekspresserer PEDF og GM-CSF, har potentiale til at forhindre retinal degeneration ved at afbøde virkningerne af oxidativ stress, hæmme inflammation og understøtte celleoverlevelse11,12,13,14,15,16 . Selvom der er flere metoder til at levere gener til celler, har vi valgt det ikke-virale hyperaktive Tornerose transposon-system til at levere PEDF- og GM-CSF-generne til RPE-celler på grund af dets sikkerhedsprofil, integrationen af generne i værtscellernes genom og dets tilbøjelighed til at integrere de leverede gener i ikke-transskriptionsaktive steder, som vi tidligere har vist17, 18,19.
Cellulær oxidativ stress kan induceres i celler, der dyrkes in vitro af flere oxidative agenser, herunder hydrogenperoxid (H2O2), 4-hydroynonenal (HNE), tertbutylhydroperoxid (tBH), høje iltspændinger og synligt lys (fuldspektret eller UV-bestråling)20,21. Høje iltspændinger og lys kræver særligt udstyr og forhold, hvilket begrænser overførselsevnen til andre systemer. Stoffer som H2O2,HNE og tBH fremkalder overlappende oxidative stress molekylære og cellulære ændringer. Vi valgte H2O2 til at teste antioxidant aktivitet PEDF og GM-CSF, fordi det er praktisk og biologisk relevant, da det er produceret af RPE celler som en reaktiv ilt mellemliggende under fotoreceptor ydre segment fagocytose22 og det findes i okulære væv in vivo23. Da oxidation af glutathion kan være delvist ansvarlig for produktionen af H2O2 i øjet, har vi analyseret niveauet af GSH / glutathion i vores undersøgelser, som er knyttet til H2O2-induceret oxidativ stress og den regenerative kapacitet af cellerne21,22. Analysen af glutathion niveauer er særligt relevant, da det deltager i de anti-oxidative beskyttelsesmekanismer i øjet24. Eksponering for H2O2 bruges ofte som model til at undersøge RPE-cellernes oxidative stressmodalitet og antioxidantaktivitet1,25,26,27,28,29,30, og derudover viser den ligheder med lysinduceret oxidativ stressskade, en "fysiologisk" kilde til oxidativ stress21.
For at evaluere funktionaliteten og effektiviteten af neuroprotektive faktorer har vi udviklet en in vitro-model, der gør det muligt for analysen at kvantificere den antioxidative effekt af vækstfaktorer udtrykt af celler, der er genetisk modificeret til at overekspressere PEDF og GM-CSF. Her viser vi, at RPE-celler transfected med generne for PEDF og GM-CSF er mere resistente over for de skadelige virkninger af H2O2 end ikke-transfected kontrolceller, som det fremgår af cellemorfologi, tæthed, levedygtighed, intracellulær niveau af glutathion og ekspression af UCP2-genet, som koder for mitokondrie upcouplingprotein 2, der har vist sig at reducere reaktive iltarter (ROS)31.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner</td>
			<td>7657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate white with clear flat bottom&#160;</td>
			<td>Costar</td>
			<td>3610</td>
			<td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning&#160;</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamid 40%</td>
			<td>Biorad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>AMIMED</td>
			<td>4-05F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-GMCSF</td>
			<td>ThermoFisher Scientific</td>
			<td>PA5-24184</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG/IgA/IgM&#160;</td>
			<td>Agilent</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-PEDF&#160;</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-390172</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-penta-His&#160;</td>
			<td>Qiagen</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-Akt</td>
			<td>Cell Signaling Technology</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-rabbit IgG H&#38;L-HRP</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody donkey anti-rabbit Alexa Fluor 594&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody goat anti-mouse Alexa 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE-19 cell line</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>chamber culture glass slides</td>
			<td>Corning</td>
			<td>354118</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTox-Glo Cytotoxicity Assay&#160;</td>
			<td>Promega</td>
			<td>G9291</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Ham`s F12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8062</td>
			<td></td>
		</tr>
		<tr>
			<td>Duo Set ELISA kit</td>
			<td>R&#38;D Systems&#160;</td>
			<td>DY215-05</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISAquant kit</td>
			<td>BioProducts MD</td>
			<td>PED613-10-Human</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyes (human)</td>
			<td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Brunschwig</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega plate reader&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software (version 8.0)</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Glo Glutathione Assay</td>
			<td>Promega</td>
			<td>V6912</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrogen peroxide (H2O2)</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software (image processing program)</td>
			<td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&#8211;2014</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol&#160;</td>
			<td>Axonlab</td>
			<td>A1378.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI4000B microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II&#160;</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SW1.5.1 software</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Axonlab</td>
			<td>3468.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Marienfeld-superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow&#160;</td>
			<td>Qiagen</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose&#160;</td>
			<td>VWR</td>
			<td>732-3197</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Lum G Gel Imaging System</td>
			<td>Aplegen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR Green FastMix</td>
			<td>Quantabio</td>
			<td>95072-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>158127-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Invitrogen&#160;</td>
			<td></td>
			<td>See Table 1 in Supplementary Materials</td>
		</tr>
		<tr>
			<td>pSB100X (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>M&#225;t&#233;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td>
		</tr>
		<tr>
			<td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>QIAGEN</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hGM-CSF&#160;</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hPEDF&#160;&#160;</td>
			<td>BioProductsMD</td>
			<td>004-096</td>
			<td></td>
		</tr>
		<tr>
			<td>ReliaPrep RNA Cell Miniprep System</td>
			<td>Promega</td>
			<td>Z6011</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Applichem</td>
			<td>A2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer system for WB&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMix qScript</td>
			<td>Quantabio</td>
			<td>95048-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15504020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>AppliChem</td>
			<td>A4975</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>AppliChem&#160;</td>
			<td>A1390</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>WesternBright ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045-D50</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman nitrocellulose membrane</td>
			<td>Chemie Brunschwig</td>
			<td>MNSC04530301</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

Oxidativ stress spiller en afgørende rolle i flere degenerative sygdomme, herunder aldersrelateret makuladegeneration (AMD), en patologi, der påvirker ~ 30 millioner patienter på verdensplan. Det fører til et fald i retinal pigment epitel (RPE)-syntetiseret neuroprotektive faktorer, f.eks pigment epitel-afledt faktor (PEDF) og granulocyte-makrofag koloni-stimulerende faktor (GM-CSF), efterfulgt af tabet af RPE celler, og i sidste ende fotoreceptor og retinal ganglion celle (RGC) død. Vi hypotese, at rekonstitution af neuroprotektive og neurogene retinale miljø ved subretinal transplantation af transfected RPE celler overekspressere PEDF og GM-CSF har potentiale til at forhindre retinal degeneration ved at afbøde virkningerne af oxidativ stress, hæmme inflammation, og støtte celle overlevelse. Ved hjælp af Tornerose transposon system (SB100X)menneskelige RPE celler er blevet transfected med PEDF og GM-CSF gener og vist stabil genintegration, langsigtet genekspression, og protein sekretion ved hjælp af qPCR, vestlige blot, ELISA, og immunofluorescence. For at bekræfte funktionaliteten og styrken af PEDF og GM-CSF udskilles af de transfected RPE celler, har vi udviklet en in vitro assay til at kvantificere reduktionen af H2O2-induceretoxidativ stress på RPE celler i kultur. Cellebeskyttelse blev evalueret ved at analysere cellemorfologi, tæthed, intracellulært niveau af glutathion, UCP2 genekspression og celle levedygtighed. Begge transfected RPE-celler, der overekspresserer PEDF og/eller GM-CSF, og celler, der ikke er transfected, men forbehandlet med PEDF og/eller GM-CSF (kommercielt tilgængelige eller renset fra transficerede celler), viste signifikant antioxidantcellebeskyttelse sammenlignet med ikke-behandlede kontroller. Den nuværende H2O2-modeler en enkel og effektiv tilgang til at evaluere antioxidanteffekten af faktorer, der kan være effektive til behandling af AMD eller lignende neurodegenerative sygdomme.

Induktion af oxidativ stress i human Retinal Pigment Epitelceller ARPE-19 og primære hRPE-celler blev behandlet med varierende koncentrationer på H2O2 i 24 timer, og antioxidantglutalsens intracellulære niveau blev kvantificeret (figur 2A,B). H2O2 ved 50 μM og 100 μM påvirkede ikke produktionen af glutathion, mens der ved 350 μM var et betydeligt fald i glutathion i ARPE-1...

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Vi præsenterer en protokol for udvikling og brug afan oxidativ stress-model ved at behandle retinale pigment epitelceller med H2O2, analysere celle morfologi, levedygtighed, tæthed, glutathion, og UCP-2 niveau. Det er en nyttig model til at undersøge antioxidanteffekten af proteiner udskilt af transposon-transfected celler til behandling af neuroretinal degeneration.

Induktion og analyse af oxidativ stress i tornerose Transposon-Transfected Human Retinal Pigment Epitelceller

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Research

JoVE Journal

Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identifikation af<em&gt; Tornerose</em&gt; Transposon Insertioner i solide tumorer med Linker-medieret PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Transkriptom analyse af menneskets retinale kirurgiske prøver vha. Journal

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Kryopræservering og dyrkning af humane amnion epitelceller ved kliniske anvendelser

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

Udførelse subretinale Injektioner i Gnavere at levere retinale pigmentepitel celler i suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Studere i bugspytkirtlen Kræft stamceller Karakteristik for udvikling af nye behandlingsstrategier

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

En trin for trin Protokol for subretinal kirurgi i kaniner

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

Udtømning af Mouse Celler fra Humane tumorxenoplantater Markant Forbedrer Downstream Analyse af Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Induktion af accelereret åreforkalkning i Mus: Den "Wire-Skade" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Fosfor-31 Magnetic Resonance Spectroscopy: et redskab til måling<em&gt; In vivo</em&gt; Mitokondrie Oxidativ phosphorylering Kapacitet i human skeletmuskulatur

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...