Manuscript:2005-11-13631B
Journal:Nature
ArticleTitle:SomaticstemcellnichetropisminWolbachia
Manyexperimentssuchastransfectionofcellsandorganisms,liveimagingandcertainbiochemicalassays,requireisolationoflive,viablebacteriafrominsects.
ThisprotocolwasdevelopedtoisolateWolbachiafromadultDrosophila,butitcanbeadaptedforotherinsects.Insomeinsectslegremovalpriortoisolationfacilitateshemolymphextrusion1.ThisisnotnecessaryforDrosophila.
Conventionally,fortransfectionexperiments,Wolbachiaareisolatedfromembryoniccytoplasm2-5.Incomparison,themainadvantagesofthismethodare:
Bettereliminationofdebrisfromultra-filtration.Faster;requireslessmanipulation.Lessyolkisco-purifiedwiththebacteria.Duetotheassociatedautofluorescence,yolkbecomesanissueforimaging(seealso“LiveimagingofWolbachia”).
1.Drosophila(orotherinsect)infectedwithWolbachia.ManystocksfromtheBloomingtonstockcenterareinfectedwithWolbachia
6.HavealsoastockfreeofWolbachiatobeusedascontrol.AWolbachiafreecontrolstockcanbeeasilyobtainedbyusingflyfoodcontainingtetracycline(0.25mg/ml),forthreegenerations.2.Ultrafree–MCCentrifugalFilterUnity.Thislowbindingmicroporousfilterassembledonamicrocentrifugetubeallowsfiltrationof
BIOLOGicalsolutions(
MilliporeCorporation,Bedford,MA).Wolbachiadiametersizesvaryfrom0.5to1.5micrometer.Twofilterssizeweretested.Inourhands,eventhesmalleronedidnotseemtoreduceyieldandproducedacleanerproduct:0.65micrometers,CatalogueNo.
UFC3ODV5.0micrometers,CatalogueNo.
UFC3OSV3.70%EtOH(v/v)4.Sterile
H201.Microcentrifuge.
30minutes.
(Useautoclavedmicrocentrifugetubesandgloves)
1-Collect50-100adultinsects(forDrosophila,femaleswillgivehigheryield).AnesthetizeonCO2.Keepthefliesand70%EtOhonice.2-TransferanesthetizedfliestotheCentrifugalFilter(5or0.65micrometer).3-Wash5timeswith70%ethanol.Invertthetubeacoupleoftimesbetweenwashes.ThisstepistocollectasmuchdebrisandsterilizetheflysurfaceasmuchaspossIBLe.4-Spinfor5-10secondsintabletopmicrocentrifugeatlowspeedsetting(around1,000g)todisposemostoftheEtOH5-Wash3xwithsterileH2O.6-Spin5-10secondstoeliminatemostofthewater7-Transferthefilterunittoasterilemicrocentrifugetube.8-Spinfor3minutesataround10,000g.Incaseyolkisnotdesired(e.g.forvisualization)lowspeedcentrifugationisrecommendedtoavoiddisruptionofeggsinsidefemales.Speedscangoaslowas1,000gfor3minutes.9-From50to100fliestheyieldisaround10-20µl.TomaximizethebacteriaviABIlity,keeponiceallthetime.Theexudatescanbeusedfortransfectionintootherhosts(see“TransfectionofWolbachiaintoadulthosts),cellcultureorimaging.
ThepresenceofbacteriacanbequicklyverifiedwithaDNAstain(see“LiveimagingofWolbachia”).Asacontrol,alwayscomparewithhemolymphisolatedfromanuninfectedstock.
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GirishDeshpandeforreviewofthismanuscript.
Wolbachiatransfectiontransinfectionhorizontaltransferartificialtransfer
