Name: isolating malignant and non-malignant b cells from lck:egfp zebrafish
Uploaded: 2019-02-22T15:00:00
Description: Watch this Scientific Journal Video about Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish at JoVE.com

Vi vill tacka Megan Malone-Perez för Zebrafiskar vård och OUHSC flöde flödescytometri kärnan. Detta arbete stöds av bidrag från Hyundai Hope på hjul, Oklahoma Center för befordran av vetenskap och teknik (HRP-067), en NIH/NIGMS INBRE pilotprojekt utmärkelse (P20 GM103447). JKF innehar den E.L. & Thelma Gaylord begåvad stol i pediatrisk hematologi-onkologi i Children's Hospital Foundation.

Alla förfaranden som inbegriper Zebrafiskar godkändes av institutionella djur vård och användning kommittén (IACUC) vid University of Oklahoma Health Sciences Center.
1. isolera icke-maligna B och T-lymfocyter från transgena lck:eGFP fisk
<ol><li>Söva fisken med hjälp av 0,02% tricaine (MS-222) i fisk systemet vatten.</li>
	<li>Undersöka 2 – 6 månad gammal fisk för fluorescerande thymi, som är belägna på den dorsomedial aspekten av gäl hålighet i ...

<ol>
	<li>Paik, E. J., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+development+in+the+zebrafish.">Hematopoietic development in the zebrafish.</a> International Journal of Developmental Biology. 54 (6-7), 1127-1137 (2010).</li><li>Kasheta, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+T+reg-like+cells+in+zebrafish.">Identification and characterization of T reg-like cells in zebrafish.</a> Journal of Experimental Medicine. 214 (12), 3519-3530 (2017).</li><li>Liu, X., 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=Zebrafish+B+Cell+Development+without+a+Pre-B+Cell+Stage,+Revealed+by+CD79+Fluorescence+Reporter+Transgenes.">Zebrafish B Cell Development without a Pre-B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes.</a> Journal of Immunology. 199 (5), 1706-1715 (2017).</li><li>Page, D. 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K., 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=Heritable+T-cell+malignancy+models+established+in+a+zebrafish+phenotypic+screen.">Heritable T-cell malignancy models established in a zebrafish phenotypic screen.</a> Leukemia. 23 (10), 1825-1835 (2009).</li><li>Rudner, L. A., 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=Shared+acquired+genomic+changes+in+zebrafish+and+human+T-ALL.">Shared acquired genomic changes in zebrafish and human T-ALL.</a> Oncogene. 30 (41), 4289-4296 (2011).</li><li>Gutierrez, A., 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=Pten+mediates+Myc+oncogene+dependence+in+a+conditional+zebrafish+model+of+T+cell+acute+lymphoblastic+leukemia.">Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia.</a> Journal of Experimental Medicine. 208 (8), 1595-1603 (2011).</li><li>Borga, C., 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=Simultaneous+B+and+T+cell+acute+lymphoblastic+leukemias+in+zebrafish+driven+by+transgenic+MYC:+implications+for+oncogenesis+and+lymphopoiesis.">Simultaneous B and T cell acute lymphoblastic leukemias in zebrafish driven by transgenic MYC: implications for oncogenesis and lymphopoiesis.</a> Leukemia. , (2018).</li><li>Haferlach, T., 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=Clinical+utility+of+microarray-based+gene+expression+profiling+in+the+diagnosis+and+subclassification+of+leukemia:+report+from+the+International+Microarray+Innovations+in+Leukemia+Study+Group.">Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group.</a> Journal of Clinical Oncology. 28 (15), 2529-2537 (2010).</li><li>Novershtern, N., 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=Densely+interconnected+transcriptional+circuits+control+cell+states+in+human+hematopoiesis.">Densely interconnected transcriptional circuits control cell states in human hematopoiesis.</a> Cell. 144 (2), 296-309 (2011).</li><li>Menke, A. L., Spitsbergen, J. M., Wolterbeek, A. P., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+the+adult+zebrafish.">Normal anatomy and histology of the adult zebrafish.</a> Toxicology Pathology. 39 (5), 759-775 (2011).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. , (2010).</li><li>Pruitt, M. M., Marin, W., Waarts, M. R., de Jong, J. L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+the+Side+Population+in+Myc-induced+T-cell+Acute+Lymphoblastic+Leukemia+in+Zebrafish.">Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish.</a> Journal of Visualized Experiments. (37), (2017).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nature Immunology. 4 (12), 1238-1246 (2003).</li><li>Carmona, S. J., 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=Single-cell+transcriptome+analysis+of+fish+immune+cells+provides+insight+into+the+evolution+of+vertebrate+immune+cell+types.">Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types.</a> Genome Research. 27 (3), 451-461 (2017).</li><li>Tang, Q., 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=Dissecting+hematopoietic+and+renal+cell+heterogeneity+in+adult+zebrafish+at+single-cell+resolution+using+RNA+sequencing.">Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.</a> Journal of Experimental Medicine. 214 (10), 2875-2887 (2017).</li></ol>

Vi utvecklat och tillhandahåller ett protokoll för att isolera B-celler från lck:eGFP transgena Zebrafiskar, att lägga detta till andra D. rerio modeller med B-lineage etiketter3,4. Något överraskande, gick identifiering av GFPlo B celler i denna linje obemärkt sedan dess beskrivning år 2004. Allmänhet, lck anses vara T-cell-specifika6, men senare studier hittade oväntade lck uttryck...

Zebrafiskar ger kraftfull attribut, till exempel genetiska manipulability, hög fruktsamhet, optisk genomskinlighet och snabb utveckling som underlättar studerar ryggradsdjur utveckling med hjälp av genetiska metoder. Dessa fördelar, tillsammans med de dela funktionerna av lever- och däggdjurspatogener blodbildningen, gör D. rerio idealisk för Invivo analyser av lymphopoiesis och lymfocyter funktion, från deras tidigaste utseende i larver under hela vuxenlivet. Blod utveckling i zebrafisk är beroende av väl bevarade genetiska processer som delas med däggdjur, och dessa omfatta det adaptiva immunsystemet. Molekylära mekanismer som styr lymfoida utveckling bevaras dessutom anmärkningsvärt mellan Zebrafiskar och däggdjur1.
Under de senaste 2 decennierna, transgena D. rerio linjer att etiketten specifika blod härstamningar och muterade bristfällig i dessa härstamningar har skapats2,3,4,5. En av dessa, lck:eGFP transgena linjen, använder Zebrafiskar lymfocyter protein tyrosine kinase (lck) arrangören för att köra GFP uttryck6. Denna gen, som uttrycks mycket av både T-lineage prekursorer och mogna T-lymfocyter, tillåter Invivo spårning av thymic T cell utveckling och ex vivo rening av T-lineage celler av FACS7. Tidigare använde vi denna linje i en framåt-genetiska ENU mutagenes skärm att identifiera könsceller mutanter benägna att T-ALL och studera somatically förvärvade genetiska händelser kopplade till T cell onkogenes8,9.
Vårt laboratorium ytterligare nyligen, nyttan av lck:eGFP Zebrafiskar. I dubbel-transgena rag2:hMYC (mänskliga MYC), lck:eGFP D. rerio som är kända för att utveckla T-ALL 10, upptäckte vi att B-lineage alla också uppstå11. Till skillnad från T-ALL i denna modell, som fluorescerar i ljust på grund av hög GFP uttryck, B-allt är svagt fluorescerande på grund av låga GFP nivåer, så att fisk med B-ALL särskiljas grovt från dem med T-ALL med hjälp av fluorescerande mikroskopi. Detta differentiella GFP uttryck tillåter också separation av GFPlo B-ALL celler från GFPHej T-ALL celler använder FACS11. Dessutom är låg lck uttryck inte unikt för Zebrafiskar B-ALL, som mänskliga B-ALL också snabb låga nivåer av LCK11,12. Jämväl, normala B-lineage celler D. rerio, möss och människor också uttrycka låga nivåer av lck/Lck/LCK, med omogna B-celler som har den högsta uttryck11,13. På grundval av per cell express B-lineage celler i lckeGFP Zebrafiskar eller derivat linjer 1-10% så mycket god Jordbrukarsed som T-lymfocyter. Dessa GFPlo celler uttryckligt kännetecken B cell mRNA som pax5 cd79b, blnk, btk, ighm, ighzoch andra, och kan renas från märg, bräss, mjälte eller perifera blod11. Därför isolerade både B - och T-lineage celler kan vara från lckeGFP Zebrafiskar, och när det gäller rag2hMYC, lckeGFP djur, B - och T-ALL celler samt11.
Här presenterar vi våra protokoll till effektivt FACS-rena icke-maligna B-celler från lck:eGFP Zebrafiskar och icke-maligna eller maligna B-celler av rag2hMYC; LCK:eGFP fisk, använder olika källa vävnader. Sådana celler kan jämväl kvantifieras av flödescytometri utan FACS isolering, om så önskas. Upptäckten av låg lck uttryck- och följaktligen låg GFP uttryck-b celler öppnar nya dörrar av experimentella möjligheter för lckeGFP Zebrafiskar, såsom i vivo B cell utvecklande studier. Således har denna transgena linje, först rapporterats i 2004, nytt liv när vi försöker utnyttja det för att snappa upp nya insikter om Zebrafiskar adaptiv immunitet.

<table>
	<tbody>
		<tr>
			<td>35 &#181;m mesh</td>
			<td>Sefar Filter technology</td>
			<td>7050-1220-000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene round-Bottom tube with cell-strainer cap</td>
			<td>Falcon Corning Brand</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>VWR international</td>
			<td>525-0448</td>
			<td></td>
		</tr>
		<tr>
			<td>AZ APO 100 Fluorescent microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoflex</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Qi1MC camara</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methansesulfonate; MS-222</td>
			<td>Sigma</td>
			<td>E-10521</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSJazz</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10.2</td>
			<td>FlowJo, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lck:eGFP</td>
			<td></td>
			<td></td>
			<td>See Langeneu et al., 2004</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>Version 4.13</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin -Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle micro-tube homogenizers</td>
			<td>Electron Microscopy Sciences</td>
			<td>64788-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Transfer pippetes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rag2:hMYC-ER</td>
			<td></td>
			<td></td>
			<td>See Gutierrez et al., 2011</td>
		</tr>
		<tr>
			<td>RPMI Media 1640 1X</td>
			<td>Life Technologies</td>
			<td>11835-030</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating malignant and non-malignant b cells from lck:egfp zebrafish

Sebrafisken (Danio rerio) är en kraftfull modell för att studera lymfocyter utveckling. Som däggdjur besitter D. rerio en adaptiva immunsystemet som innehåller B- och T-lymfocyter. Studier av zebrafisk lymphopoiesis är svårt eftersom antikroppar erkänner D. rerio cell yta markörer inte är i allmänhet tillgängliga, komplicerande isolering och karakterisering av olika lymfocyter befolkningen, inklusive B-härstamning celler. Transgena linjerna med härstamning-specifika fluorophore uttryck används ofta för att kringgå denna utmaning. Raden transgena lck:eGFP har använts för att studera D. rerio T cell utveckling, och har också använts till modell T-cellernas utveckling och akut lymfatisk leukemi (T-ALL). Även om lck:eGFP fisk har använts att analysera T-härstamning, har de inte använts för att studera B-celler. Nyligen upptäckte vi att många Zebrafiskar B-celler uttrycker också lck, om än på lägre nivåer. Följaktligen uttrycker lck:eGFP B celler jämväl låga nivåer av GFP. Utifrån detta konstaterande har utvecklat vi ett protokoll för att rena B-lineage celler från lck:eGFP Zebrafiskar, som vi redovisar här. Vår metod beskriver hur att utnyttja en fluorescerande-aktiverad cell sorterare (FACS) för att rena B-celler från lck:eGFP fisk eller relaterade rader, till exempel dubbel-transgena rag2:hMYC; LCK:eGFP fisk. I dessa rader, B-celler, särskilt omogna B-celler, express GFP på låg men detekterbara nivåer, så att de kan särskiljas från T-celler, som uttrycker GFP högt. B-celler kan isoleras från märg, bräss, mjälte, blod eller andra vävnader. Detta protokoll ger en ny metod för att rena D. rerio B-celler, som möjliggör studier som fokuserar på ämnen som B cell utveckling och B-lymfocyter maligniteter.

Vi använde flödescytometri att analysera och FACS att isolera GFPlo och god JordbrukarsedHej celler från thymus, njure benmärg och mjälte från lck:eGFP transgena Zebrafiskar. Analys av 3-månad-gammal fisk visade bräss innehöll mestadels GFP+ lymfocyter. GFP+ celler var i stort sett begränsade till lymfoida gaten tidigare beskrivits av Traver et al.17. Två distinkta GFP+ populationer, GFP<sup...

Watch this Scientific Journal Video about Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish at JoVE.com

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgena lck:eGFP Sebrafisken uttrycker GFP högt i T-lymfocyter, och har använts för att studera T cell utveckling och akut lymfatisk leukemi. Denna linje kan användas till studie B-celler, som uttrycker lck på lägre nivåer. Det här protokollet beskriver rening av maligna och icke-maligna B-celler från lck:eGFP Zebrafiskar.

Isolera elakartat och icke-elakartat B-celler från lck:eGFP Zebrafiskar

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

Vi fann nyligen att många B-celler i LCK GFP zebrafisk uttrycka låga nivåer av GFP. Detta gör denna linje ett användbart verktyg för att studera B-celler samt T-celler. Den största fördelen med denna teknik är att nu kan vi studera B-celler och T-celler från LCK GFP zebrafisk eller så kan vi studera B-ALL och T-ALL celler från fiskar med cancer.Dessa metoder möjliggör B-cellutveckling och B-ALL-studier inom zebrafisk. Som är relevanta för att förstå det adaptiva immunsystemet och det är maligniteter vanliga i barncancer. Om du aldrig har gjort dessa dissektioner innan du kanske vill förfina din teknik några gånger tills du kan få bra bräss och märg prover.Visuell demonstration kommer att hjälpa tittarna att känna igen fluorösa och B-ALL i fisk och att välja rätt inställningar för diskriminerande B och T-celler för deras isolering. Efter anesthetizing med 0,02%Trikain i fisk system vatten använda GFP-filter på en epifluorescence mikroskop för att undersöka två till sex månader gamla zebrafish för fluorescerande thymi vid den dorsala mediala aspekten av greniala hålighet. Efter att lokalisera brässen placera avlivade fisk i en Petri-skålen och använda fluorescerande mikroskop för att ta bort GFP positiva thymi.Och brightfield mikroskopi inställningar för att skörda njurmärgen och mjälte. Placera varje samlat organ i en 1,5 milliliter rör som innehåller 500 mikroliter av kallt sortering medium och använda en mortelstöt microtube homogenisator för att homogenisera vävnaderna på is. Sedan, stam den homogeniserade vävnaden genom en 35 mikrometer mesh filter för att generera en enda cell suspension och placera cellerna på is.Börjar vid två till fyra månader använda ett fluorescerande mikroskop för att skärmen dubbel-transgenic rag2:hMYC LCK EGFP fisk med låg exponering inställningar för att identifiera ljusa T-cells akut lymfatisk leukemi eller T-ALL och hög exponering inställningar för att identifiera dim B-ALLA djur. Vild typ kontroll och pre-leukemic fisk har GFP lokaliserade endast i bräss. Kategorisera fisken baserat på omfattningen av GFP fluorescens med hjälp av en enkel tre kategori system.Nivå ett fisk visa en fluorescerande signal som en thymic tumör med endast en begränsad lokal spridning. Nivå två fisk visa en fluorescerande signal bortom bräss som omfattar mindre än 50%av kroppen. Och nivå tre fisk visa en fluorescerande signal som sträcker sig bortom 50%av kroppen.Separera fisken med ALLA från dem utan cancer. Och övervaka pre-leukemic fisk utan GFP positiva tumörer en gång varje månad för utveckling av nya ALLA. För T eller B-ALL cell isolering använda ett rakblad för att ta bort huvudet och thymic regionen av en avlivas nivå tre fisk och placera kroppen i en petriskål.Använd en P-1000-pipetten för att tvätta fiskhinnehålan med 500 mikroliter av kallt sorteringsmedium, samla in cellerna och medium i ett fem milliliterrör. Med hjälp av en färsk pipettspets injicera två till tre ytterligare 200 till 300 mikroliter volymer av kallt sorteringsmedium i kroppen hålighet och använda spetsen av pipetten för att tillämpa försiktigt tryck på fiskkroppen att pressa ut cellerna ur kroppen hålighet i fem milliliter röret. Sedan, stam cellen suspension genom en 35 microliter mesh filter.Och placera cellerna på is tills deras analys genom flödescytometri. För hela kroppen homogenisering använd en mortelstöt microtube homogenisator för att homogenisera kroppen av fisken och lägga till ytterligare 300 mikroliter av kall sortering medium till röret. Filtrera cellupphängningen genom ett 35 mikrometernätfilter, tvätta filtret med extra sorteringsmedium efter behov tills endast vävnadskräp finns kvar på filtret.Placera sedan cellerna på is tills deras analys. För flödescytmetrisk analys av de isolerade cellerna ställer flödescytometerparametrarna enligt tillverkarens riktlinjer. Och förvärva 10, 000 till 50, 000 händelser för att ställa in den framåt scatter och sida scatter parametrar för att definiera lymfocyt och progenitor cellgrindar och att utesluta cellulära skräp.Uteslut celldubbel och använd lymfoida och/eller prekursorgrindarna för att bestämma antalet och procentandelen av de GFP-positiva cellerna. Använda phycoerythrin och GFP-intensiteterna definiera grindar för GFP negativa kontra GFP låg kontra GFP höga celler. Att lära sig var man ska placera portarna för att isolera GFP-låg- och GFP-höga celler är avgörande för att få rena B- och T-cellpopulationer.Två distinkta GFP positiva populationer, GFP låga B-celler och GFP höga T-celler kan erhållas från brässet. B-celler som är bosatta i njure märgen av tre månader gamla zebrafish uttrycka låga nivåer av GFP. GFP höga celler är knappa dock, vilket tyder på att endast en liten andel av T-lymfocyter är närvarande i märgen vid tre månaders ålder.Likaså splenic prover visar högre procentsatser av GFP låg än GFP höga celler. I dubbel-transgena fiskar som ännu inte har utvecklat akut lymfatisk leukemi, GFP uttryck i bräss, märg och mjälte liknar den som observerats i enkel-transgenic LCK EGFP fisk. Antalet lymfocyter per organ ökas dock.Förmodligen, på grund av MYC driven expansion av omogna B och T-cell populationer där rag2 promotorn är aktiv. I dubbel-transgenic fisk med B och eller T-ALL som har utvecklat fluorescerande cancer med sex månader B-ALL är svagt fluorescerande cancer som innehåller mestadels GFP låga celler. Däremot kan ljust fluorescerande fisk hysa antingen isolerade T-ALL eller blandade populationer av både GFP låg B-ALL och GFP höga T-ALL-celler.I lydningen, som bedömts av kvantitativ analys av polymeraskedjan, GFP låga celler uttrycker högre nivåer av B-cellavskrifter. Och GFP höga celler uttrycker högre nivåer av T-cellgener, LCK och GFP-markören. Det är viktigt att lära sig att känna igen fisk med B-ALL genom mikroskopi.Och hur man korrekt definiera flödet cytometri grindar för att separera GFP låg och GFP höga populationer. Efter denna procedur kan du isolera DNA, RNA, eller protein från GFP låg och GFP höga celler för PCR, sekvensering, vad som finns i blod eller in vivo transplantation experiment. När vi lärt oss LCK GFB fisk har fluorescerande B-celler utöver T-celler vi kunde studera båda celltyperna från samma djur att utvärdera B och T-ALL.

isolating-malignant-and-non-malignant-b-cells-from-lckegfp-zebrafish

Research

JoVE Journal

Medicine

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Orthotopic Xenografting mänskliga luciferas-taggade Malignt Perifera tumör Nerve Slida celler för In vivo Testning av kandidat behandlingsmetoder

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolering av mänskliga Islets från Delvis Pancreatectomized Patienter

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Mame Modeller för 4D Live-cell imaging av tumör: mikromiljö Interaktioner som påverkar malign progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolering, karakterisering och jämförande differentiering av mänskliga Dental celler Pulp Stem Härstammar från permanenta tänder med hjälp av två olika metoder

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tredimensionell kulturer: Ett verktyg för att studera Normal Acinar arkitektur kontra malignt Omformning av bröstceller

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Metod för att erhålla primär äggstockscancerceller från Solid Prover

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studera bukspottkörtelcancer Stem Cell egenskaper för att utveckla nya behandlingsstrategier

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

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Isolering och förökning av cirkulerande tumörceller från en mus Cancer

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Förkalkning av vaskulära glatta muskelceller och avbildning av aorta förkalkning och inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolering och profilering av MicroRNA innehåller exosomes från Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...