1.Mediumwithsupplements(serum,glutamine,antibiotics). 2.STE,37oC.Storedat4oC. 3.Trypsin,0.25%or0.05%,37oC.Storedat-20oC.Donotleavetrypsininwaterbathforanextendedperiodoftime. 4.Pipets:10ml,5ml(optional),Pasteurpipets.Pipetingaid. 5.Miscellaneousitems:pipetingaids,trayforusedglassware,Sharpie.Seegeneralguidelinesforcellculture. 6.Freshculturedishes. 1.Setupnecessaryitemsinthehood.Spraytheexteriorsurfaceofcontainerswith70%alcohol.Alsosterilizeyourhands. 2.Removeallmediumfromthestockdish. 3.AddSTE,~5mlfor100mmdish,~3mlfor60mmdish.Rinsetheentiresurfacebyrockingthedish. 4.RemoveSTE. 5.Add~1mltrypsinwithaPasteurpipet.Rockthedishgentlyandremoveallthetrypsinrightaway(usingthesamepipet). 6.Setupnewdishesandaddanappropriateamountofmedium,~10mlfor100mmdishes,~4mlfor60mmdishes,~2-2.5mlformicroinjectiondishes. 7.Checkcellsunderthescope.Add2-5mlmediumwhenalmostallcellsareround.Donotleavecellsintrypsinmuchlongerthannecessary. 8.Gentlyblowcellsoffthesurfaceofthedish.Rotatethedishtorecovercellsfromtheentiresurface. 9.AddanappropriatevolumeofthecellsUSPensiontothefreshdishes.Gentlyrock/swirlthedishtospreadoutthecells. 10.Cleanupthehoodandthevacuumsuctionline.SubculturingCells
