细胞培养1

CellCultureandCellIsolationBasicTechniquesforMammalianCellTissueCultureCellIsolationRuijunZhangInstituteofImmunologyinUSTCBasicTechniquesforMammalianCellTissueCultureCellsubculturingCellfreezingCellrecoveringCellcountingCellsubculturingTrypsinizingandSubculturingCellsFromaMonolayerPassagingCellsinSuspensionCulturesPROCESSRemoveallmediumWashingcellswithCa2+Mg2+-freePBSTrypsin/EDTAsolutiontodislodgecellsWashcellsandcountthecellsnumberCompletemediumtoinhibittheTrypsinactivityAddfreshmediumtoculturecellsNoteCa2+andMg2+inthesaltsolutioncancausecellstosticktogetherandinhibitthetrypsinactivityInhibitthefurthertrypsinactivitytoprotectthecellsPROCESSSwirlflasktosuspendthecellsRemovecellsuspensionAddfreshmediumtoculturecellsSplitthecellsWashingcellsCellfreezingTheprocessThenotePROCESSHarvestthecellswithtrypsinWashingcellsResuspendthecellswithfreezingmediumTransferthecellsintoliquidnitrogenstoragefreezerAdjustthecellsconcentrationof106~107/mlGradualtemperaturedropHarvestthecellsCellsfromsuspensionCellsfrommonolayerNoteFreezingmedium:completemediumsupplementedwith30%-50%(v/v)FBSand10%(v/v)DMSOGradualtemperaturedropKeepouttobecontaminatedBesuretosuspendthecellpelletcarefullyCellrecoveringTheprocessThenotePROCESSRemovevialfromliquidnitrogenfreezerImmediatelyplaceitintoa37°CwaterbathTransfercellsintoasterilecentrifugetubeCheckculturesafter24hWashingcellsAddfreshmediumtocellpelletandcultureCellsfromsuspensionCellsfrommonolayerNoteCareshouldbetakennottospillliquidnitrogenontheskin.Themediumshouldbethawedasquicklyaspossible.Alternativetemperature42C.CellcountingTheprocessCellactivitydeterminingThenotePROCESSLoadhemacytometerCellcountingCellnumbercalculatingCellssuspensionpreparingHemacytometerPreparingHemacytometerNoteAllowcellstosettleforafewminutesbeforebeginningtocount.CountcellstouchingthemiddlelineofthetriplelineonthetopandleftofthesquaresAmaximumcellcountof20to50cellsper1´1-mmsquareisrecommended.cells/ml=averagecountpersquare´dilutionfactor´104totalcells=cells/ml´totaloriginalvolumeofcellsuspensionDeterminecellviabilitybystainingwithtrypanLoadhemacytometerCellcountingCellnumbercalculatingCellssuspensionpreparingHemacytometerPreparingStaincell0.4%trypanfor5minPreparationFibroblastPreparationandCultureofHumanLymphocytesCellIsolationPROCESSPrepareskinsampleRemovesubcutaneoustissueRemoveepidermisCutthesampleintosmallsquaresPlacesomeskinpiecesintissueculturedishPlaceglasscoverslipgentlyovertheskinspecimensAddfreshmediumandculturecellsSubculturecellsUponconfluencyFreezecellsEpidermisRemovingCutthehumanskinsamples:Incubate45minto4hrwith0.5%dispase/PBSina37CwaterbathIncubatewith0.3%trypsin/PBSfor30to60minina37Cwaterbathorovernightat4CPlacethesamplewiththeepidermalsideupandscrapeofftheepidermismechanicallyusingtwopairsofforcepsNoteItiscrucialtouseanew,finesurgicalscalpeltocutthespecimensforfibroblastoutgrowthoccursonlyfromsharplycutedges.DonotsweepawaytheoverslipIsolationofHumanLymphocytesPreparationofperipheralbloodmononuclearcells(PBMC)byFicoll-hypaquecentrifugationPreparationoflympnocytesfromPBMCPROCESSDilutethewholebloodwithEDTA/PBSUsingroomtemperatureFicoll-HypaquesolutionunderlayincentrifugetubeAddgentlydilutedbloodonthesurfaceofFicoll-HypaquesolutionCentrifuge20minat800gAspiratetheinterfacebandWashingcellsNoteDonotbreaktheinterfacebetweenbloodandFicoll-HypaquesolutionPROCESSIsolationPBMCbydensitygradientcentrifugationAddPBMCintotheplastictissueculturedishIncubate3hr,gentlyrockingflaskseveryhourHarvestnonadherentcellsAddfreshmediumandculturecells