AliquotingMatrigel:

Dayone:

Putthesterilizedtipbox(either200mlor1000mltips),sterilizedEppendorftubecontainer,andappropriatepipettorin-20°Cfreezer.

ThawtheMatrigelbottleoniceinthe4°Cfridgeovernight(untilitliquifies).

Daytwo:

EachbottleofMatrigelisatadifferentdensity*,anditisaliquotedoutat2mg/tube(whichisenoughtocoatonesix-wellplate).

FillanicebucketandplacetheMatrigel,alongwithpipetsandtubesonicetokeepthemcold**.

ExamplecalculationwithMatrigelarrivingat13.841mg/ml:

13.841mg/1ml=2mg/xml

x=0.145ml/tube

Usinggoodsteriletechnique,aliquotMatrigeltotubes,switchingtipswheneverMatrigelseemstobecloggingthetipand/orcausingthepipettomeasureinaccurately.Placetubesoniceastheyarefinished.

Whentheentirebottlehasbeenaliquotedthetubesarestoredineitherthe–20°Cor–70°Cuntilreadytobeused.

HowtoMakeMatrigelPlates:

ThereareatleasttwopossIBLemethodsforthis.Themainobjectiveisnottolettheundilutedmatrigelsitatroomtemperaturefortoolong(oritwillbecomechunkyandsolidify).

Thawtubeovernightoniceat4°C.

Dilutewith6mlcoldbasalmediaandmixwell.

Add1mlperwellof6wellplate.

Allowplatetositatroomtemperatureforonehourorovernightat4°C.

Platemayeitherbeusedimmediatelyorstoredat4°C(platewillbegoodforatleastoneweek).

Whenreadytousetheplate(s),removetheexcessliquidandwashoncewithbasalmedium.

Asecondpossiblemethodisalittlequicker,theonlymodificationbeingthatyoutakeatubedirectlyfromthefreezerandIMMEDIATELYresUSPendthepelletofMatrigelin6mlicecoldmedia.Keeppipettingvigorouslyuntilallchunksaregone,andaddtoplate.

Ifaftereitherofthesemethods,yourplatestilllookslikeithaschunksofMatrigelinsteadofasmoothevenlayer,tryplacingtheplateat4°Covernight.Thisshould"re-melt"thematrigelintoanevenlayer.Somechunksareacceptable,butmakesurethatthewholesurfaceoftheplateiscoatedwithmatrigel.

CaringforCellsGrowingonMatrigel:

hEScellsgrownonMatrigel-coatedplatesrequireMEF-conditionedmedium.ThismediumisidenticaltoregularhESmedium,butismadewithoutbFGF.Twenty-fourhoursbeforeusingthemedium,itisconditionedonMEFs(atadensityof2.12x105cells/ml,2.5ml/wellof6wellplate).bFGFisaddedfresheverytimetoeachindividualwell(5ml/well).

*WhenorderingMatrigel,itisbesttorequest"high-density"Matrigel(ie.over12mg/ml).Therearesomeapplicationsinthelabwhichrequireahigherdensitytohavesuccessfulexperiments.Additionally,onebottleofhighdensityMatrigelwillgofartherthanonebottleoflowdensityMatrigel.

**ThemainobjectiveandconstantthroughoutworkingwithMatrigelistokeepitCOLD.AsMatrigelwarmstoroomtemperature,itbeginstosolidify,makingithardertoworkwith(ie.stickinginsideofthepipet)andmakingitchunkierwhenattemptingtouseandplate.