注：感谢哈佛大学医学院果蝇RNAi筛选中心的支持和提供本实验方法

I.MEDIUM

A.S2,S2C,S2*,S2R+,S3,l(2)mbn,Kc(167),&DL2cells:

Schneider"s/10%FBS/PS450mlSchneider"smedium(Gibco#11720-034)(pouroff50mlintoFalcontosaveasserum-free)***[S2R+andKccellswillnotgrowinSchneider"smediumfromSigma]50mlFetalBovineSerum(JRH#12103-78P)-HeatInactivated(aliquotedin-20)5ml1:100Penicillin-Streptomycin(Gibco#15070-063)(aliquotedin-20)

Sterilefilter(0.2µ)Storeat4^oC-donotfreeze

B.ML-DmBG2&ML-DmBG6cells:

M3/10%FBS/PS/Insulin450mlShieldsandSangM3insectmedium(Sigma#S8523)(pouroff50mlintoFalcontosaveasserum-free)50mlFetalBovineSerum(JRH#12103-78P)-HeatInactivated(aliquotedin-20)5ml1:100Penicillin-Streptomycin(Gibco#15070-063)(aliquotedin-20)10ug/mlInsulin(Sigma#I6634)

Sterilefilter(0.2µ)Storeat4^oC-donotfreeze

C.clone8(CL8)cells:

CompleteM3MediumShieldsandSangM3insectmedium(Sigma#S3652)2%FetalBovineSerum(JRH#12103-78P)-HeatInactivated(aliquotedin-20)2.5%Flyextract(seebelow)0.0125IU/mlInsulin(Sigma#I6634)1xPenicillin-Streptomycin(Gibco#15070-063)(aliquotedin-20)

Sterilefilter(0.2µ)

HeatInactivatingFetalBovineSerum

1. ThawtheFetalBovineSerumonashakerat2-8oC(overnight)
2. Pre-heatawaterbathtoatemperatureof56oC.(Makesurethewatercoversalloftheseruminthebottle)
3. Heattheserumfor30minutesinthe56oCbath.
4. Allowserumtocooltoroomtemperaturebeforeaddingtocells.

InsulinStock(100x)Dissolve10mg(25IU)in0.5mlof0.01NHCL.Heatin37^oCtodissolveforfewminutes.Add19.5mLofM3Mediatobringvolumeupto20mL.FilterSterilizewitha0.22µmfilter.Alliquotandstorestockat-20^oC.Workingstockmaybekeptat4^oCfor4-5weeks.

FlyExtract

1. Collectabout30gofhealthyflies.200fliesweighabout0.22g,use1.5mlM3mediumfor0.22g.Tomake200mlhomogenate,use30gofflies.(7.5ml/1g).
2. Placefliesinfreezerfor~20".Removefliesfromfreezerandweighout1/2ofthemandputrestbackinfreezer.AddtheappropriateamountofM3medium.
3. Placefliesplusmediuminablender.Blendatmediumspeeduntilitlooksasifalltheflieshavebeenlysedandthesupernateisreddishfromtheeyepigments.[Oldmethod:Remove5mloftheflysUSPensionandcrushinadouncehomogenizer(whichshouldbeextremelyclean)untiltheplungerreachesthebottomofthehomogenizer.Collecttheresultantmushin50mlconicaltubes.Repeatuntilallfliesarehomogenized.]
4. Transferlysateto50mlconicaltubesandspinat2600rpminatabletopcentrifugeatRT.Removethesupernateandtransfertoanewtube.
5. Removetheoilytoplayer.
6. Heatinactivatetheextractina60^oCwaterbathfor10".Youwillseeaprecipitateform.
7. Centrifugeat2600rpmfor1houratRT.Removesupernateandsterilizethrough0.22µmfilter.Youwillgothroughseveralfilters,soeitheruseaprefilter,orbepreparedtowastealotofprepackagedfilters.
8. Storein12.5mlaliquots(2.5%finalin500ml)andkeepat-20^oCafterfreezinginLN₂.

II.GROWTHCONDITIONS

Cellsgrow@23-25^oCand@RTDonotneedCO²Splitevery3-4daystomaintainDensitysensitive-dieiftoodenseortoodilute

III.MAINTENANCE

Splitoneflaskofcells(mostrecentdate)intotwonewT75flasks(VWR#BD353136)whencultureisconfluent.Keepotherflaskasabackup.Monitorgrowthstatusbymicroscopybeforesplittinganddecidewhethertoadjustrecommendeddilutionfactoraccordingly.

A.Semi-adherentcelllines-willsticktonewflaskbutcomelooseovertime

B.Adherentcelllines

SL2,S2C,S2*

DetachcellsfromtheflaskeitherbybangingorscrapingPipetcellsupanddownabout10timestoresuspendcellsandseparateclumpsAliquotcellsintonewflasksaccordingly

DL2,DL1,S2R+,Kc167,Clone8,S3

Protocol1:RemoveallmediumScrapecellswithscraperResuspendcellswith10mLoffreshmedium,pipettingupanddownabout10timesAliquotcellsintonewflasksaccordingly

Protocol2:RemovemediumWashinPBStoremoveanyserumAdd5mltrypsin;letsit5-10"Bangcellsoffflask.Add5mlserummediumtoinactivatetrypsinandwashremainingcellsoffbottomofflask.Spindowncellsat1200-1400rpmfor5min.Resuspendcellsinserummedium.Aliquotcellsintonewflasksaccordingly

Protocol3:DetachcellsfromtheflaskeitherbybangingorscrapingPipetcellsupanddownabout10timestoresuspendcellsandseparateclumpsAliquotcellsintonewflasksaccordingly

BG2

Removemedium(saveandfilterthroughsyringefilterforuseas"conditionedmedium").WashinPBStoremoveanyserumAdd5mltrypsin;letsit5-10"Bangcellsoffflask.Add5mlserummediumtoinactivatetrypsinandwashremainingcellsoffbottomofflask.Spindowncellsat1200-1400rpmfor5min.Resuspendcellsinconditionedmedium.Aliquotcellsintonewflasksaccordingly

CanalsouseAccutaseinsteadoftrypsin.

IV.FREEZING

Growcellstosubconfluencey-approximately1-2x10⁷cells/ml.Labelappropriate#ofcryovials.Removecellsfromflask(trypsinizeandresuspendinmediumifnecessary).Countcells.Spincellsat1200rpm5"Aspirateoffmedium.Resuspendatapproximately1.1x10⁷cells/mlinfreezingmedium:FBS+10%DMSO,filteredORCompletemedium+10%DMSO,filteredAliquot1mlcellsuspension/cryovial.Putvialsinfoambox,tapeclosed,andplacein-70^oC.Afterafewdays,transfervialstoLN₂forlongtermstorage.

V.THAWING

Prepare15mlconicaltubewith5mlmedium.Thawcellsquicklyinwaterbath.Justbeforecellsarecompletelythawed,decontamiatetheoutsideofthetubewith70%EtOH.Transferthecellstotheconicaltubewith5mlmedium.Spinat1200rpm5".Aspirateandresuspendcellsin5mlmedium.PlateinaT25flask.Watchdaily.Initially,cellsmayneedtobesplitatirregularintervals.

VI.COUNTING

Remove~0.5mlcellsfromflask(trypsinizecellsifnecessary)intoEppendorf.DilutecellsintoTrypanBlueaccordingtoestimateddensity.(confluentculturestry30:70µlto50:50µlofcells:TB).Transfer10-12µlcellstohaemocytometer.Countcellsinoppositecorners(~100-200/largesquare).Calcuation:(count#1+count#2)/2x(dilutionfactor)x10⁴=Xcells/ml(usually~1-10x10⁷)