Name: live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy
Uploaded: 2014-06-24T14:00:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

We would like to thank Dr. Alengo Nyamay'antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.

1. Isolering av råtta neonatal cardiomyocytes <ol><li> Använd Dulbeccos modifierade Eagles medium (DMEM) och minimalt essentiellt medium (MEM) kompletterat med fetalt bovint serum (FBS) och penicillin / streptomycin (P / S) som odlingsmedium. Lägg bromodeoxiuridin (BrdU) till mediet för att förhindra tillväxt av fibroblaster för de första två dagarna efter isoleringen. </li></ol><table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Basmedium </td><td> FBS </td><td> BrdU (mM) </td><td> P /...

<ol>
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Primära kardiomyocyter isolerade från neonatala råttor har länge använts för att studera cardiomyocyte funktioner in vitro. Detta protokoll beskriver en metod för isolering av neonatala hjärtmuskelceller från råttungar genom att använda en två-stegs enzymdigere metoden först digerering med trypsin O / N vid 4 ° C och därefter renat kollagenas. En fördel med användning av renat kollagenas steget är att samma grad av enzym används för alla isoleringar. Således är kvaliteten och mängden av i...

Primära råttneonatala hjärtmuskelceller har länge använts för att undersöka cardiomyocyte funktion in vitro 1. De är lätta att isolera från råttungar av flera väl etablerade metoder 2-4. Den vanligaste metoden använder kollagenas eller trypsin för att smälta bindväv av hjärtat före cellisolering. Forskare har också utvecklat metoder för att isolera hjärtmuskelceller från vuxna gnagare 5-8 samt neonatala möss 9,10. Detta protokoll beskriver en metod för isolering av kardiomyocyter från neonatala råttungar, som utnyttjar en två-stegs enzymdigere procedur. Trypsin används först O / N vid 4 ° C, och sedan renade kollagenas vid 37 ° C. Inkubation av hjärtvävnad med trypsin O / N vid 4 ° C reducerar nödvändiga åtgärder för att skörda celler jämfört med metoder som använder sekventiella inkubationer i en varm enzymlösning 2. Dessutom, genom användning av renat collagenase snarare än råa enzymer, kan mycket-till-lot variation elimineras, vilket ger förbättrad reproducerbarhet. Funktionella studier av ett särskilt protein har man ofta en proteinexpressionssystem med användning av ett adenovirus 11 till 13 och / eller ett lentivirus 14-16. [Varnande anmärkning] Den virala produktion och manipulation bör utföras enligt NIH riktlinjer. Adenovirus integrerar inte in i värdgenomet. Den har en mycket hög effektivitet av transduktion i de flesta celltyper, inklusive delande celler och icke-delande celler, såväl som primära celler och etablerade cell-linjer. Detta gör adenovirus en tillförlitlig vektor för genuttryck. Höga nivåer av det protein som kodas av adenovirusvektorn utvecklas inom 48 timmar efter transduktion, och de kan pågå i flera veckor 17. Men en nackdel med att använda ett adenovirus för proteinexpression är att utvecklingen av en rekombinant adenovirus är både komplicerat och tidskrävande. Denna nackdel förklarar delvis varför många forskare har vänt sig till lentiviruses för rekombinant genuttryck. Till skillnad adenovirala konstruktioner, genererar en lentiviral konstruktion är snabbt och enkelt. Även lentiviruses har generellt lägre effektivitet på transduktion än adenovirus, både delande och icke-delande celler, de integreras i värdgenomet. Följaktligen är uttryck för den omvandlade genen stabilare för lentiviruses än för adenovirus. På grund av tekniska framsteg inom området mikroskopi, är det mycket lättare att fånga levande cell bilder av celler som uttrycker rekombinanta proteiner. Detta gäller även vid videohastighet hastigheter på förvärv. Detta möjliggör för forskaren att bestämma hur vissa förändringar i proteinet av intresse funktionellt påverka cellen i realtid. Den konfokala spinning disk mikroskop har flera viktiga funktioner som gör det en optimal teknik för live cell imaging 18,19. Den Yokogawa snurrande skiva möjliggör en mycket snabbare bildtagning, medan samtidigt utnyttjar mycket mindre lasereffekt än punkt scanning konfokala mikroskop. Båda dessa särdrag är på grund av den roterande skiva, som innehåller ett flertal konfokala hål genom vilket lasern passerar samtidigt till proverna. Vid förvärv, själva disken snurrar snabbt och kontinuerligt 18-20. Genom att använda autofokussystemet i mikroskopet, är stabil fokus bibehålls under långa tidsperioder. Detta gör det möjligt för forskare att ta väl fokuserade levande cellbilder. Förvärvade bilderna spelas upp som en filmfil. Bilderna analyseras med hjälp av bildanalys program som ImageJ 21,22, FIJI 23, eller annan kommersiellt tillgänglig programvara.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1 scissors for decapitation</td>
			<td>WPI</td>
			<td>501749</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>1 fine scissors for heart isolation and chopping</td>
			<td>WPI</td>
			<td>14393</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>2 fine forceps (Dumont No. 5)</td>
			<td>Sigma</td>
			<td>F6521</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Three sterilized 10 cm plastic dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for hearts isolation</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative.</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-0.17-14-C</td>
			<td>for TIRF or high resolution image</td>
		</tr>
		<tr>
			<td>Ethanol solution, 70 % (v/v) in water</td>
			<td>Sigma</td>
			<td>E7148</td>
			<td></td>
		</tr>
		<tr>
			<td>2% gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>One sterilized 10 cm plastic dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for trypsinization</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>parafilm</td>
			<td>Sigma</td>
			<td>P7543</td>
			<td></td>
		</tr>
		<tr>
			<td>Two 10 cm plastic cell culture dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for selection</td>
		</tr>
		<tr>
			<td>Auto pipette</td>
			<td>Drummond Scientific&#160;</td>
			<td>4-000-300</td>
			<td>for trituration</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Cellometer, automated cell counter</td>
			<td>nexcelom</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>&#160; Microscope and Hematocytometer</td>
			<td>any brand</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>invitrogen</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-19AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>incubating orbital shaker</td>
			<td>Sigma</td>
			<td>Z673129</td>
			<td>to shake heart tissue with collagenase at 37 C at 170-200 rpm</td>
		</tr>
		<tr>
			<td>10 mg/ml BrdU solution</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>invitrogen</td>
			<td>41965039</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>invitrogen</td>
			<td>31095029</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)&#160;</td>
			<td>invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 2 lentiviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>three packaging plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>The lentiviral transfer vector, pLVX-IRES-Puro</td>
			<td>Clontech</td>
			<td>632183</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (serum-free medium)</td>
			<td>invitrogen</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>transfection reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; polyethyleneimine&#8220;Max&#8221;, (Mw 40,000) - High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form)&#160;</td>
			<td>Polysciences</td>
			<td>24765-2</td>
			<td>It can be substituted with X-tremeGENE 9 from Roche</td>
		</tr>
		<tr>
			<td>&#160; X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365779001</td>
			<td>substitute for PEI as transfection reagent</td>
		</tr>
		<tr>
			<td>chloroquine</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td>dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 &#956;M Chloroquine.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Luer-Lok syringe, sterilized</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um filters</td>
			<td>Sigma</td>
			<td>F8677</td>
			<td>use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus.</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>dissolve in water and make 8mg/ml stock solution, then filter it to sterilize.</td>
		</tr>
		<tr>
			<td>polyethylene glicol 6000</td>
			<td>Sigma</td>
			<td>81260</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 3 Adenoviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Some 10 cm cell culture dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplate with lid, flat-bottom, tissue culture, sterile</td>
			<td>BD Falcon</td>
			<td>353072</td>
			<td>for titration</td>
		</tr>
		<tr>
			<td>Multichannel piptte, 10-100 ul, 8-channel</td>
			<td>eppendorf</td>
			<td>3122 000.035</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 4 live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal microspopy</td>
			<td>PerkinElmer</td>
			<td>L7267000</td>
			<td></td>
		</tr>
		<tr>
			<td>a temperature controlled chamber</td>
			<td>any brand</td>
			<td></td>
			<td>to keep temperature at 37 C</td>
		</tr>
		<tr>
			<td>a CO2 environmental system</td>
			<td>any brand</td>
			<td></td>
			<td>optional to maintain CO2 concentration optimal</td>
		</tr>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; CO2 Independent Medium, No Glutamine</td>
			<td>invitrogen</td>
			<td>18045-054&#160;&#160;</td>
			<td>for long time time-lapse imaging</td>
		</tr>
		<tr>
			<td>&#160; DMEM, High Glucose, HEPES, no Phenol Red&#160;</td>
			<td>invitrogen</td>
			<td>21063-029</td>
			<td>for long time time-lapse imaging</td>
		</tr>
	</tbody>
</table>

live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy

Primär råtta neonatal cardiomyocytes är användbara i grundläggande in vitro kardiovaskulär forskning, eftersom de lätt kan isoleras i stort antal i ett enda förfarande. På grund av framsteg inom mikroskopteknik är det relativt enkelt att fånga levande cellbilder i syfte att undersöka cellulära händelser i realtid med minimal oro angående fototoxicitet till cellerna. Detta protokoll beskriver hur man tar levande cell Timelapse bilder av primär råtta neonatal cardiomyocytes med hjälp av en konfokala spinning disk mikroskop efter lentiviral och adenoviral transduktion för att modulera egenskaper i cellen. Tillämpningen av två olika typer av virus gör det lättare att uppnå en lämplig transduktion ränta och uttrycksnivåer för två olika gener. Väl fokuserade levande cellbilder kan erhållas med hjälp av mikroskop s autofokussystem, som upprätthåller stabil fokus under långa tidsperioder. Att tillämpa denna metod, funktioner exogent ingenjöred proteiner som uttrycks i odlade primära cellerna kan analyseras. Dessutom kan detta system användas för att undersöka funktionen av gener genom användning av siRNA liksom av kemiska modulatorer.

För att illustrera tekniken, ett lentivirus kodning EGFP (förbättrat grönt fluorescerande protein)-tagged Cx43 (Connexin43) eller en muterad Cx43 32 användes för att uttrycka EGFP-taggade proteiner i celler, och ett adenovirus kodande FGFR1DN (fibroblast growth factor receptor 1 dominant negativ ) användes för att stänga av FGF-signalering i cellen 33-35. Tre dagar efter isolering av hjärtmuskelceller, var de isolerade cardiomyocytes omvandlas med lentivirus för att uttrycka EGFP-taggade...

Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Levande cell imaging av Primär Rat Neonatal Cardiomyocytes Efter Adenoviral och Lentiviral Transduction Använda Konfokal Spinning Disk mikroskopi

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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Biology

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