Purpose

Todescribethemaintenanceofcellsinculture.

Safety

Equipment

Laminarflowhood

CO2Incubator

MechanicalPipettor

Invertedmicroscope

Vacuumpumpandflask

Materials

Completecellculturemedium,appropriateforthecellline

Tissuecultureflasksofappropriatesizes

Tissuecultureplates-96wellor24well.

Sterilepipets,assortedsizes

Multichannelpipetandsteriletips.

Pasteurpipets,9?±,sterile,unplugged.

70%alcohol

SterilePetridishes.

Procedure

Generalconsiderations

Turnonthehoodandallowtorunforatleast10minutesbeforestarting.

Pre-warmallmediain37oCwaterbath.

Wipeallsurfaceswith70%alcoholbeforestarting.

Forcellsinflasks.

Cellsaresplitorrefedevery3-4days.

Removetheflasksfromtheincubator.Examineflaskswithinvertedmicroscope.

Placeflasksandmediumunderhood.

RemoveanaliquotofthecellsUSPensionforcountingwithahemocytometer(SP05-009)andviABIlitydetermination(SP09-005).

Labeltheappropriatenumberofnewflaskswiththecelllinename,thepassage,theslitratioorseedingdensityandthedate.

Asepticallytransfertherequirednumberofcellstothenewflask.Addfreshmediumtotheflask.

ForT-25flask-maximumof10mlofmedium.

ForT-75flask-maximumof50mlofmedium.

ForT-150flask-maximumof100mlofmedium.

ForT-225flask-maximumof200mlofmedium.

Recaptheflask(s),gentlyshaketoevenlydispersethecells.

Returntheflaskstotheincubator.Loosenthecaps1/2turn,ifnecessary.

For96wellplates.

Platesarere-fedevery3-4days.

Removetheplatesfromtheincubator,andexaminemicroscopicallyorwiththemirror.Markwellsthatarepositiveforcellgrowth.

Placeplatesandmediumunderthehood.

Ifsupesarebeingcollectedforanassay,pre-labeltheplatesforthesupernatants.

Transfer150mlofsupernatanttotheclean,labeledplate.

Storeallsupernatantplatesat2-80Cuntilreadytoassay.

RemovespentmediumwithasterilePasteurpipetattachedtoavacuum.Holdthepipetata45degreeangleabout1/2waydownthewell.

Startatthetopleftcorneroftheplateandworkbackandforthacrossthewellstothelowerrightcorner.

Repeatwithallplates.

AsepticallytransfermediumintosterilePetridish.Add150-175mloffreshmediumintoeachwellusingamultichannelpipettor.Workfromtheendoftheplatefurthestfromyourhand,tominimizethenumberoftimesyourhandpassesovertheplate.

Repeatwithallplates.Addadditionalmediumtoanew,cleanPetridishifmoreisneeded.Changepipettipsforeachplate.

Returntheplatestotheincubator.

For24wellplates

Removeplatesfromtheincubatorandexaminemicroscopically.

Placeplatesandmediumunderthehood.

Ifsupesarebeingcollectedforanassay,pre-labelstheplatesforthesupernatants.

Transfer1-2mlofsupernatanttotheclean,labeledplate.

Storeallsupernatantplatesat2-8oCuntilreadytoassay.

RemovespentmediumwithasterilePasteurpipetattachedtoavacuum.Holdthepipetata45degreeangleabout1/2waydownthewell.

Startatthetopleftcorneroftheplateandworkbackandforthacrossthewellstothelowerrightcorner.

Repeatwithallplates.

Ifcellsneedtobesplit,indicatedbyorangecoloredmediumandthecellscoveringmorethanhalfthesurfaceofthewell,add2mloffreshmediumtothewell.

Resuspendthecellsbygentlypipetting.

Transfer0.5mlofcellsuspensiontoeachnewwell.

Add2mloffreshmediumtoeachwell.

Returntheplatestotheincubator.

Transferringcellsfroma96to24wellplate.

Identifytheclonestobeexpanded.

Usingasterilepipettipandpipettor,resuspendthecellsbypipetting.

Transfer150mlofthecellsuspensiononewellofapre-labeled24wellplate.

NOTE:Useonlythetoprowofeach24wellplatefornewclonestoallowroomtoexpandthecellsdowntheplateastheygrow.

Add2mloffreshmediumtoeachwellofthe24wellplate.Add150mloffreshmediumtoeach96well.