Name: conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment
Uploaded: 2017-11-23T14:00:00
Description: Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

Forfatterne vil gerne anerkende Jacqueline Rosenberg for korrekturlæsning håndskriftet. Dette arbejde blev støttet af den os Department of Health and Human Services/National Institutes of Health med et tilskud til YA DeClerck (Giv 5R01 CA 129377) og TJ Martell Foundation. MH Kubala er modtageren af en forskning karriere udvikling Fellowship tildeling af det Saban forskningsinstitut på children's Hospital Los Angeles.

Afsnittet protokol, der anvender humane monocytter fra raske frivillige følger retningslinjerne fra children's Hospital Los Angeles menneskelige forskning etiske komité og er blevet godkendt af den institutionelle Review Board under den menneskelige materiale Protokol nummer: CCI 08-00208.
1. forberedelse af kræft cellelinjer med tetracyklin-regulerede shRNA udtryk
<ol><li>Kloning af shRNA PAI-1 i Tet-pLKO-puro vektor 8 , <sup class="xre...

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Sammensat af en række forskellige celletyper, er TME afgørende for udviklingen af kræft. For at sikre optimale vækstbetingelser, tiltrække kræftceller monocytter gennem udskillelsen af kemotaktisk faktorer. Her præsenterer vi en protokol for at studere mekanismen af monocyt rekruttering af tumor celler i vitro. Til dette formål anvendes en kombination af inducerbar gen expression system, rensning af primære humane monocytter og som et eksempel på en migration assay, Boyden kammer assay.
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Tumor forbundet makrofager (TAMs) bidrage til tumor udvikling ved at fremme tumorvækst, metastaser og regulere immunrespons2. Kræftceller rekruttere inflammatoriske monocytter, der infiltrerer tumoren og differentiere sig til pro-tumorfremkaldende TAMs3. Infiltration af tumor med TAMs korrelerer med dårlig klinisk resultat og har været knyttet til rollen immunosuppressive makrofager4,5. Men mekanismer for rekruttering af makrofager at tumor er ikke godt udforsket og en bedre forståelse af de involverede veje er afgørende for videre avancement felt og lovende behandlinger. En af udfordringerne i at studere samspillet mellem kræftceller og normale celler i tumor mikromiljø (TME) er kompleksiteten af de mekanismer og celler involveret, som kræver in vitro- metoder, der tillader dissektion af krydstale. Her præsenterer vi en alsidig metode, der kan anvendes til at undersøge den paracrine virkning af en kræft celle-afledte, udskilles protein migration af andre celletyper som makrofager, in vitro. Bruger et system hvor udtrykket af shRNA mod en kræft celle-afledte protein involveret i ansættelse af monocytter er under kontrol af tetracyklin inducerbar promotor, er paracrine effekten af udskilles protein på monocytter quantitated. I denne protokol efterfulgt kloning af shRNA sekvenser til tetracyklin regulerede vektor præsenteres af generation af stabil kræft cellelinjer. Yderligere, rensning af primære humane monocytter og en Boyden kammer analysen bruges til at analysere paracrine effekten af en kræft-celle afledte protein migration af monocytter.
Downregulation af protein kodende gener er almindeligt anvendt med siRNA og shRNA teknikker, dog ikke uden begrænsninger i dets anvendelse. Den langsigtede knock-down (KD) af gener kan fremkalde sekundære adaptive responser af celler, der interfererer med eksperimentelle resultater. Manglende tidsmæssig kontrol over genekspression gør det udfordrende at studere et protein dynamisk rolle over tid eller rollen som et protein, der er afgørende for celle overlevelse. Dette spørgsmål er især vigtigt i i vivo indstillinger, hvor rollen som et protein i tumor udvikling og progression kan kræve downregulation af udtryk for protein af interesse kun efter at tumor er etableret. Betinget KD har fordelen at forhindre en for tidligt dødelige virkning af KD på celler og for at aktivere analyse af rollen, som proteinet i forskellige stadier af tumorvækst, mens ubetinget KD kunne resultere i manglende tumor udvikling.
En række betinget KD systemer er blevet udviklet for at afhjælpe begrænsningerne af stabil KD. Betinget gen expression systemer omfatter Ecdysone-inducerbar overekspression systemer, cytokrom P-450 induktion system1og tetracyklin reguleret gen expression systemer. Tetracyclin-reguleret gen expression systemer giver kontrol over udtryk for shRNA ved tilsætning af antibiotikum tetracyklin (eller dens mere stabil analog - doxycyclin). I Tet-ON systemer, udtryk for shRNA er induceret i overværelse af tetracyklin/doxycyclin resulterer i en genekspression KD, mens i Tet-OFF systemer, udtryk for shRNA er undertrykt i overværelse af tetracyklin resulterer i genekspression. En ulempe ved tetracyklin-inducerbar system er tidligere rapporteret lavt niveau af udtryk for shRNA i mangel af doxycyclin - såkaldte leakiness6,7. I Tet-ON beskrevet system her, efter administration af tetracyclin, binding af constitutively udtrykt tetracyklin repressor (TetR) protein til Tet-responderende element (TRE) rækkefølgen inden for H1 promotor-regionen shRNA af interesse er undertrykt. Dette resulterer i udtryk for shRNA og hæmning af oversættelse af protein af interesse i en tetracyklin-afhængige måde8,9.
Andre tilgængelige Tet-ON systemer omfatter samtidige knock-i af Millie sekvens mellem TATA boks og proksimale sekvens element (PSE) og TATA boks og transskription starte hjemmesiden er udviklet af Chan mfl. 10 dette system kræver mindre end toksiske doser af tetracyklin at regulere shRNA udtryk, dog under en ikke-induceret stat, lave niveauer af shRNA udtrykkes. Krüppel-associerede boks (KRAB) baseret Tet-ON system11 omfatter KRAB, en finger protein, zink, som fag gener ligger inden for en 3 kb række KRAB bindingssted transcriptional undertrykkelse. Kimære protein tTRKRAB kan binde til Millie, og på grund af den store vifte af DNA styrbar kapacitet, Millie ikke skal begrænses mellem transkription starte site og initiativtageren og har lav effekt på aktiviteten af initiativtageren. Under ikke-induceret staten, blev kontrollerbar RNA interferens systemet rapporteret til at vise et lavere niveau af rende udtryk for shRNA11,12; Det kræver dog en sekventiel, to-vektor kloning tilgang. I forhold til tidligere udviklet betinget KD systemer såsom Ecdysone-inducerbar overekspression system eller cytokrom P-450 induktion system, tetracyklin-reguleret system har fordelen af dens robusthed og reversibilitet, og derfor er mest anvendte rutinemæssigt system13. Systemet anvendes i denne protokol har fordel over dobbelt Millie knock-in system og KRAB Tet-ON system som det kræver ligetil, enkelt vektor kloning, giver mulighed for hurtig generation af flere kloner, og det udstiller meget lave niveauer af leakiness i den fravær af doxycyclin.
TME er afgørende for udviklingen af kræft. For at lette tumorvækst, rekruttere kræftceller inflammatoriske monocytter ved at udskille kemotaktisk proteiner. Rekrutterede monocytter infiltrere tumor og differentiere sig til pro-tumorfremkaldende TAMs, der bidrager til tumorvækst og metastaser. In vitro undersøgelser af immun celle rekruttering udnytte migration assays, med Boyden kammer assay er almindeligt brugt14,15,16,17. I denne analyse, er chemoattractant proteinkilde, for eksempel, kræftceller, eller et oprenset protein, placeret i bunden kammer. Immunceller er placeret i den øverste kammer adskilt med porøse membran fra bunden rum. Celler migrerer mod den stigende graduering af chemoattractant, og dem, der findes på den nederste side af membranen er farves og tælles under mikroskop. Her tester vi funktionen chemoattractant af Plasminogen aktivator Inhibitor 1 (PAI-1) på monocytter ved at generere stabile, inducerbar kræft cellelinjer hvor udtryk af PAI-1 er reguleret ved doxycyclin. Vi bruger menneskelige primære monocytter i Boyden kammer migration assay for at vurdere PAI-1 rolle i monocyt migration. Forskelle mellem menneskelige primære monocytter og udbredte monocytic cellelinjer som THP1 er blevet rapporteret og omfatter forskellige cytokin udtryk mønstre18; for eksempel 5-10 fold stigning i TNF-α udtryk niveauer af THP1 celler i forhold til humane monocytter19. THP1 celler er afledt af menneskelige monocytic leukæmiceller, er nemme at vedligeholde, og formere sig med en gennemsnitlig fordobling tid af 19-50 h20. Tværtimod er humane monocytter karakteriseret ved en kort levetid i mangel af vækstfaktorer. Da monocytter er renset fra blod af donorer, en rimelig mængde af variabilitet opstår blandt personerne og afhængigt af metoden rensning forekomme forurening med andre celletyper. Ikke desto mindre primære monocytter er relevante og det er blevet anbefalet at bruge eller bekræfte de opnåede resultater med monocytic celle linjerne ved hjælp af primære monocytter i biologiske forskning19. Her beskriver vi en protokol til rensning af menneskelige primære monocytter fra perifert blod. Alternative metoder til monocyt rensning omfatte tæthed farveforløb og vedhæftning protokoller og to-trins procedure med enkelt forløb af Ficoll-Hypaque efterfulgt af en Percoll gradient21. Renhed af befolkningens monocyt fremstillet ved disse metoder varierer mellem 70-90%. Metoden beskrevet her bruger en tæthed farveforløb efterfulgt af en negativ immun udvalg22 og muliggør rensning af humane monocytter uden direkte kontakt med antistoffer, derved undgår deres utilsigtet aktivering og resulterer i en > 95% ren population af monocytter.
Protokollen præsenteres her bruges til at konfigurere en funktionel Tet-ON system for genekspression KD i menneskelige cancer cellelinjer til at undersøge chemoattractant virkningen af kræft-afledte udskilles protein PAI-1 på monocytter. PAI-1 er overexpressed af en bred vifte af tumorer og sit udtryk paradoksalt nok korrelerer med dårlig klinisk resultat23,24. Den pro-tumorfremkaldende rolle af PAI-1 er et resultat af sine pro-angiogene og anti-apoptotiske fungerer25,26. PAI-1 har vist sig at bidrage til betændelse ved at fremme rekrutteringen af makrofager til stedet for betændelse27. PAI-1 blev vist sig at fremme glatte muskulatur cellmigration28,29 og til at deltage i Mac-1 afhængig af makrofag migration30. PAI-1 overekspression har også vist sig at signifikant øge rekrutteringen af Raw 264.7 makrofager ind B16F10 melanom tumorer31. PAI-1 rolle i TAM migration har imidlertid ikke været undersøgt i detaljer. Vi bruger den beskrevne protokol til at besvare spørgsmålet om, hvorvidt PAI-1 tiltrækker monocytter til kræftceller. Denne metode giver dissektion af krydstale mellem tumor og TME ved hæmning af udskilles protein i kræftceller og analysere komponenterne af TME.

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tet-pLKO-puro lentiviral vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">21915</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS (Tetracycline-free)</td>
			<td style="width:177px;">Omega Scientific</td>
			<td style="width:119px;">FB-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque</td>
			<td style="width:177px;">Sigma</td>
			<td>10771-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human Monocyte Enrichment Kit</td>
			<td style="width:177px;">StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Magnet</td>
			<td style="width:177px;">StemCell</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgeI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3552S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cut Smart buffer</td>
			<td style="width:177px;">NEB</td>
			<td>B7200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:177px;">Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROMEGA Wizard SV Gel and PCR Cleanup System</td>
			<td style="width:177px;">PROMEGA</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:405px;">One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hema 3 stain set for Wright-Giemsa stain</td>
			<td style="width:177px;">Protocol</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293 cells</td>
			<td style="width:177px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XhoI restriction enzyme</td>
			<td style="width:177px;">NEB</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase buffer</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EDTA 0.5 M solution</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Lipofectamine 2000</td>
			<td style="width:177px;">Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">The Big Easy&#34; EasySep Magnet</td>
			<td style="width:177px;">Stem Cell</td>
			<td style="width:119px;">18001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.1% Poly-L-Lysine solution</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Acetate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium butyrate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;M syringe filters</td>
			<td style="width:177px;">VWR International</td>
			<td>28145-479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride(NaCl)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653-5 kg</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dithioerythritol (DTE)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B8255-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol (EtOH)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">792780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tryptone</td>
			<td style="width:177px;">Amresco</td>
			<td style="width:119px;">J859-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td style="width:177px;">Fluka</td>
			<td style="width:119px;">70161-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:177px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ampicillin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline hyclate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-25g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.5 M EDTA pH 8.0</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Falcon Permeable Support for 24 Well Plate with 8.0&#956;m Transparent PET Membrane</td>
			<td style="width:177px;">Becton Dickinson</td>
			<td style="width:119px;">353097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Serum Albumin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton-Tipped Applicator&#160;</td>
			<td style="width:177px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol Hema 3 STAT pack&#160;</td>
			<td style="width:177px;">Fisher Scientific Company</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resolve Immersion Oil, High Viscosity</td>
			<td style="width:177px;">Richard Allan Scientific</td>
			<td style="width:119px;">M4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Penicillin Streptomycin</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
	</tbody>
</table>

conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment

siRNA og shRNA-medieret banke ned (KD) metoder til regulering af genekspression er uvurderlige værktøjer for at forstå gen- og protein funktion. I det tilfælde, at KD protein af interesse har en dødelig effekt på celler eller den forventede effekt af KD er er tidsafhængig, ubetinget KD metoder ikke passende. Betinget systemer er mere velegnede i disse tilfælde og har været genstand for stor interesse. Disse omfatter Ecdysone-inducerbar overekspression systemer, cytokrom P-450 induktion system1, og tetracyklin reguleret gen expression systemer.
Tetracyklin reguleret gen expression system muliggør reversible kontrol over protein udtryk ved induktion af shRNA udtryk i overværelse af tetracyclin. I denne protokol præsenterer vi en eksperimenterende design ved hjælp af funktionelle Tet-ON system i menneskelige cancer cellelinjer til betinget regulering af genekspression. Vi så vise brugen af dette system i studiet af tumor celle-monocyt interaktion.

Tre shRNA sekvenser blev testet for den mest effektive KD PAI-1. For dette, blev shRNA sekvenser mod PAI-1 og scrambled (tabel 1) klonet i Tet-pLKO-puro udtryk vektor efter den protokol, der er beskrevet ovenfor. HT-1080 fibrosarcoma cellelinje var stabilt transfekteret med genererede konstruktioner og cellerne blev behandlet med doxycyklin i 3 dage. Udtryk af PAI-1 blev bekræftet ved Western blotting ()figur 2A) og intensit...

Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Denne protokol fungerer som en ordning for oprettelse af en funktionel Tet-ON system i kræft cellelinjer og dens efterfølgende anvendelse, navnlig for at studere rollen af tumor celle-afledte proteiner i rekruttering af monocytter/makrofager at tumor mikromiljø.

Betinget Knockdown af genekspression i kræft cellelinjer til at studere ansættelse af monocytter/makrofager at Tumor mikromiljø

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Research

JoVE Journal

Cancer Research

Lung Tumor Cell Recruitment Assay

Lunge Tumor celle rekruttering Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Det<em&gt; Drosophila</em&gt; Imaginære Disc Tumor Model: Visualisering og Kvantificering af genekspression og tumorindtrængen Brug Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Påvisning af mitokondrier membran potentiale til at studere CLIC4 Knockdown-induceret HN4 celle apoptose In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR vurdering af pH, pO2, Redox Status, og koncentrationen af fosfat og glutathion i Tumor mikromiljø

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

Virus levering af CRISPR Guides til Murine prostata for gen ændring

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Live-celle Imaging Assays til undersøgelse glioblastom hjerne Tumor stamcelle Migration og Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Oprettelse af cellelinjer overekspression DR3 for at vurdere den apoptotiske reaktion på anti-mitotiske Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Analyse af tumor Genekspressions faktorer med CorExplorer-webportalen

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Analysis of Side Population in Solid Tumor Cell Lines

Analyse af sidepopulation i faste tumorcellelinjer

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Kombineret betinget knockdown og tilpasset sfære dannelse assay at studere en stemness-associerede gene af patient-afledte gastrisk kræft stamceller

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...