GENERALMAINTENANCEINSTRUCTIONSFORIMAGINALDISCCELLLINES Thisisourlabprotocolforgrowingimaginaldisccells(clone-8) TheMilnerLabwebsitehasadditionalprotocolsforclone-8cells. Clone8cellsarepassagedevery3-4daysfromconfluentT25flasks(about5x107cells):splitroughly1/5to1/10.Neverallowculturestobecometoodensefortheywilldierapidly!If95%aredeadthedayafteryousplitthemyouhaveprobablyletthemgrowtoodense.Forthesamereason,alwaysusesubconfluentcellsforbiochemistryorRNAisolations.Thesecellsaretightlyadherent,especiallywhentheyhavebeenpassagedrecently.Washthecellswith3mloftrypsin,priortotrypsinization.Trypsinizeforabout1minatroomtemperature.Taptheflasksrealhardtodetachcells,immediatelyadd5mlofmediumtoinactivatethetrypsin.Spindowncellsat1200rpminatabletopcentrifugeandresUSPendtorequireddensity.Alwaysuseafreshflaskwhentransferringcells.CellscanbefrozenincompleteM3medium+10%FCSand10%DMSO;againusesubconfluentcultures. Placefliesinfreezerfor20min.Place30goffrozenfliesand200mlofmediumintoablender."Puree"inblenderapproximately2-3minutes. Spinmushat3000rpminatabletopcentrifugefor20min. Removesupernatant(leavingexoskeletonsandeyepigment)andtransfertoanewtube.Removeoilylayerontop.Heatinactivatein60Cwaterbathfor30min.Youwillseeaprecipitateform.Centrifugeat3000rpmfor30min.Transfersupernatanttonewtubeandspinagainfor15min.Removesupernatantandsterilizethrougha0.22umfilter.Storein12.5mlaliquots(2.5%finalin500ml)andkeepat-20C. 2.5%flyextract 5ug/mlinsulin 1/2xpenstrep(100xbottle=5000ug/ml)MAKINGFLYEXTRACTFORWINGDISCCELLMAINTENANCE
(CullenandMilner,TISSUEANDCELL,199123(1):29-39.)Startwithacollectionofabout30gofhealthyflies.200fliesweigh0.22g.Use1.5mlM3mediumper0.22g;tomake200mlhomogenate,use30gofflies.REAGENTS
ThecompleteM3mediumhasthefollowingadditives.
2%FBSAddinginsulin.
Insulin(Sigma,I-6634)Dissolve5mginsulinin0.3mlof0.01NHCleachtimemediumismade.AddM3mediumto5ml.Add2.5mlinsulinsolutionto500mlM3medium.Sterilizecompletemediumthrougha0.22umfilter.Trypsin
GibcoBRLTrypsin-EDTA.05%Catalog#25300-054PBS
Sigma#D5927M3Medium
ShieldsandSangsM3medium,Sigma(#S3652)FBS(heatinactivatedtestedforinsectculture),Sigma(#F3018)TRANSFECTIONS
ImaginalwingdisccelllinescanbetransfectedusingtheconventionalCa++phosphatecoprecipitationmethod.Cellsshouldbesplit1:4thedaypriortothetransfectionfromasubconfluentT75flaskinto10cmdishes.CellsareleftwiththeDNAcoprecipitateovernight.ThenextdaytheprecipitatesshouldbewashedawaywithPBS,trypsinsizedandtransferredtoacleandish.Thecellsshouldbeallowedtorecoveronedaybeforedrugselectionisstarted.Transientsalsoworkwellinthesecells.Fordrugselection250ug/mlhygromycinseemstogivegoodresultsalthoughearlyonduringtheselectionsomedrugresistantcoloniesdoappear.Coloniesshouldappearinabout14days.Whenthecoloniesconsistofafewhundredcellstheycanbescrapedoffthedishwithasterileyellowtipandtransferredtoa12welldish.Usuallyafteranother2weeksorsothesecanbetransferredtoaT25flask.
