Live-Cell Imaging Primary Rat Neonatal cardiomyocytes Efter Adenoviral og Lentiviral Transduktion Brug Confocal Spinning Disk Microscopy
Summary
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.
Abstract
Primær rotte neonatal cardiomyocytter er nyttige i grundlæggende in vitro kardiovaskulær forskning, fordi de let kan isoleres i stort antal i en enkelt procedure. På grund af fremskridt i mikroskop-teknologi er det relativt nemt at fange levende celle billeder med henblik på at undersøge cellulære begivenheder i realtid med minimal bekymring vedrørende fototoxicitet til cellerne. Denne protokol beskriver hvordan man tager levende celle timelapse billeder af primær rotte neonatale cardiomyocytter ved anvendelse af et konfokalt roterende skive mikroskop efter lentiviral og adenoviral transduktion at modulere egenskaberne af cellen. Anvendelsen af to forskellige typer af virus gør det lettere at opnå en passende transduktion rente og udtryk niveauer i to forskellige gener. Godt fokuseret levende celle billeder kan opnås under anvendelse af mikroskopets autofokus-system, som opretholder stabile fokus i lange perioder. Anvendelsen af denne metode, funktioner eksogent ingeniøred proteiner udtrykt i dyrkede primære celler kan analyseres. Derudover kan dette system anvendes til at undersøge funktionen af gener ved anvendelse af siRNAs samt kemiske modulatorer.
Introduction
Primære rotte neonatale cardiomyocytter har længe været anvendt til at undersøge cardiomyocyte funktion in vitro 1. De er nemme at isolere fra rotteunger af flere veletablerede metoder 2-4. Den mest almindelige fremgangsmåde anvender kollagenase eller trypsin til at fordøje bindevæv af hjertet før celleisolering. Forskere har også udviklet fremgangsmåder til isolering cardiomyocytter fra voksne gnavere 5-8 samt neonatale mus 9,10.
Denne protokol beskriver en fremgangsmåde til isolering af cardiomyocytter fra neonatale rotteunger, der anvender en totrins-enzymfordøjelsen procedure. Trypsin bruges først O / N ved 4 ° C og derefter renset collagenase ved 37 ° C. Inkubation af hjertevævet med trypsin O / N ved 4 ° C reducerer de nødvendige høst celler skridt i forhold til fremgangsmåder, der anvender sekventielle inkubationer i en varm enzymopløsning 2. Desuden, ved anvendelse af oprenset collagenase snarere end rå enzymer, kan parti-til-parti variation elimineres, hvilket giver forbedret reproducerbarhed.
Funktionelle undersøgelser af et bestemt protein ofte anvender et protein ekspressionssystem ved hjælp af en adenovirus 11-13 og / eller et lentivirus 14-16. [Advarende note] Den virale produktion og manipulation bør udføres i henhold til NIH retningslinjer.
Adenovirus er ikke integreret i værtsgenomet. Det har en meget høj effektivitet af transduktion i de fleste celletyper, herunder delende celler og ikke-delende celler, såvel som primære celler og etablerede cellelinjer. Dette gør adenovirus en pålidelig vektor til genekspression. Høje niveauer af proteinet kodet af adenovirusvektoren udvikle sig inden for 48 timer efter transduktion, og de kan vare i flere uger 17. Men en ulempe ved et adenovirus til proteinekspression er, at udviklingen af en rekombinant adenovirus er både kompliceret og tidskrævende. Denne ulempe forklarer til dels, hvorfor mange forskere har været at dreje til lentivira for rekombinant genekspression. I modsætning adenovirale konstruktioner, er hurtig og let at generere en lentiviral konstruktion. Selv lentivira generelt har lavere effektivitetsgevinster af transduktion end adenovirus, både delende og ikke-delende celler, de integreres i værtens genom. Følgelig ekspression af det transducerede gen er mere stabilt for lentivirus end for adenovira.
Grund af teknologiske fremskridt inden for mikroskopi, er det meget nemmere at fange levende celle billeder af celler, der udtrykker rekombinante proteiner. Dette gælder også på video rate hastigheder på erhvervelsen. Dette gør det muligt for undersøgeren at bestemme, hvordan bestemte ændringer i proteinet af interesse funktionelt påvirke cellen i realtid. Den konfokal spinning disk mikroskop har flere vigtige funktioner, der gør det til en optimal teknik til live cell imaging 18,19. Yokogawa spinning disk giver mulighed for langt mere erhvervelse hurtig billedet, mens på samme tid udnytte langt mindre laserenergi end punkt konfokal mikroskoper. Begge disse særlige funktioner er på grund af den roterende skive, som indeholder talrige konfokale huller, gennem hvilke laseren passerer samtidigt til prøverne. Under købet selve harddisken spinder hurtigt og kontinuerligt 18-20. Ved at bruge autofokus-system af mikroskopet, er stabil fokus fastholdes over lange perioder. Dette gør det muligt for forskerne at tage velfokuserede levende celle billeder. Erhvervede billeder afspilles som en film fil. Billederne er analyseret ved hjælp af billedanalyse software som ImageJ 21,22, Fiji 23 eller anden kommercielt tilgængelig software.
Protocol
Representative Results
Discussion
Primære cardiomyocytter isoleret fra neonatale rotter har længe været anvendt til at undersøge cardiomyocyte funktioner in vitro. Denne protokol beskriver en fremgangsmåde til isolering af neonatale cardiomyocytter fra rotteunger under anvendelse af en totrins-enzym fordoejelsesmetoden først fordøjelse med trypsin O / N ved 4 ° C og derefter renset collagenase. En fordel ved at ansætte den rensede collagenase skridt er, at den samme kvalitet af enzym bruges til alle isoleringer. Således er kvaliteten …
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 1 scissors for decapitation | WPI | 501749 | Autoclave before use |
| 1 fine scissors for heart isolation and chopping | WPI | 14393 | Autoclave before use |
| 2 fine forceps (Dumont No. 5) | Sigma | F6521 | Autoclave before use |
| Three sterilized 10 cm plastic dishes | Sigma | CLS430165 | for hearts isolation |
| 3.5 cm glass bottom culture dishes | MatTek | P35G-1.5-20-C | for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative. |
| 3.5 cm glass bottom culture dishes | MatTek | P35G-0.17-14-C | for TIRF or high resolution image |
| Ethanol solution, 70 % (v/v) in water | Sigma | E7148 | |
| 2% gelatin | Sigma | G1393 | |
| One sterilized 10 cm plastic dish | Sigma | CLS430165 | for trypsinization |
| Aluminium foil | any brand | ||
| parafilm | Sigma | P7543 | |
| Two 10 cm plastic cell culture dish | Sigma | CLS430165 | for selection |
| Auto pipette | Drummond Scientific | 4-000-300 | for trituration |
| Cell counter | |||
| Cellometer, automated cell counter | nexcelom | to check and count cells | |
| Microscope and Hematocytometer | any brand | to check and count cells | |
| Trypan Blue Solution, 0.4% | invitrogen | 15250-061 | |
| CO2 incubator | Sanyo | MCO-19AIC | |
| incubating orbital shaker | Sigma | Z673129 | to shake heart tissue with collagenase at 37 C at 170-200 rpm |
| 10 mg/ml BrdU solution | BD Pharmingen | 550891 | |
| DMEM, High Glucose | invitrogen | 41965039 | Mix medium as indicated in the protocol and warm before use |
| MEM | invitrogen | 31095029 | Mix medium as indicated in the protocol and warm before use |
| Fetal Bovine Serum | invitrogen | 26140079 | |
| Penicillin-Streptomycin (10,000 U/mL) | invitrogen | 15140-122 | |
| Section 2 lentiviral transduction | |||
| three packaging plasmids | |||
| pMDLg/pRRE | Addgene | 12251 | |
| pRSV-Rev | Addgene | 12253 | |
| pMD2.G | Addgene | 12259 | |
| The lentiviral transfer vector, pLVX-IRES-Puro | Clontech | 632183 | |
| Opti-MEM (serum-free medium) | invitrogen | 31985070 | |
| transfection reagent | |||
| polyethyleneimine“Max”, (Mw 40,000) – High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form) | Polysciences | 24765-2 | It can be substituted with X-tremeGENE 9 from Roche |
| X-tremeGENE 9 | Roche | 6365779001 | substitute for PEI as transfection reagent |
| chloroquine | Sigma | C6628 | dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 μM Chloroquine. |
| HEPES | Sigma | H3375 | |
| 10 ml Luer-Lok syringe, sterilized | BD | 309604 | |
| 0.45 um filters | Sigma | F8677 | 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. |
| Hexadimethrine bromide | Sigma | H9268 | dissolve in water and make 8mg/ml stock solution, then filter it to sterilize. |
| polyethylene glicol 6000 | Sigma | 81260 | |
| Sodium chloride | Sigma | S9888 | |
| sodium hydroxide | Sigma | S5881 | |
| Section 3 Adenoviral transduction | |||
| HEK 293T cells | ATCC | CRL-11268 | |
| Some 10 cm cell culture dishes | Sigma | CLS430165 | |
| 96-Well Microplate with lid, flat-bottom, tissue culture, sterile | BD Falcon | 353072 | for titration |
| Multichannel piptte, 10-100 ul, 8-channel | eppendorf | 3122 000.035 | |
| Section 4 live cell imaging | |||
| Spinning disk confocal microspopy | PerkinElmer | L7267000 | |
| a temperature controlled chamber | any brand | to keep temperature at 37 C | |
| a CO2 environmental system | any brand | optional to maintain CO2 concentration optimal | |
| Medium | |||
| CO2 Independent Medium, No Glutamine | invitrogen | 18045-054 | for long time time-lapse imaging |
| DMEM, High Glucose, HEPES, no Phenol Red | invitrogen | 21063-029 | for long time time-lapse imaging |
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