SubculturingCells

1.Mediumwithsupplements(serum,glutamine,antibiotics).

2.STE,37^oC.Storedat4^oC.

3.Trypsin,0.25%or0.05%,37^oC.Storedat-20^oC.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.

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