AimManyculturesobtainedfromaculturecollection,suchasECACC,willarrivefrozenandinordertousethemthecellsmustbethawedandputintoculture.ItisvitaltothawcellscorrectlyinordertomaintaintheviABIlityofthecultureandenabletheculturetorecovermorequickly.Somecryoprotectants,suchasDMSO(Prod.No.D2650),aretoxicabove4ºCthereforeitisessentialthatculturesarethawedquicklyanddilutedinculturemediumtominimizethetoxiceffects.

Materials

• Media–pre-warmedtotheappropriatetemperature(refertotheECACCCellLineDataSheetforthecorrectmediumandsizeofflasktoresuscitationinto.)
• 70%ethanolinwater(Prod.No.R8382)
• DMSO(Prod.No.D2650)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat,safetyvisor)
• Waterbathsettoappropriatetemperature
• MicroBIOLOGicalsafetycabinetatappropriatecontainmentlevel
• CO₂incubator
• Prelabeledflasks
• MarkerPen
• Pipettes
• AmpuleRack
• Tissue

Procedure

1. ReadTechnicaldatasheettoestablishspecificrequirementsforyourcellline.
2. Preparetheflasksasappropriate(informationontechnicaldatasheet).Labelwithcelllinename,passagenumberanddate.
3. Collectampuleofcellsfromliquidnitrogenstoragewearingappropriateprotectiveequipmentandtransfertolaboratoryinasealedcontainer.
4. Stillwearingprotectiveclothing,removeampulefromcontainerandplaceinawaterbathatanappropriatetemperatureforyourcelllinee.g.37ºCformammaliancells.Submergeonlythelowerhalfoftheampule.Allowtothawuntilasmallamountoficeremainsinthevial-usually1-2minutes.TransfertoclassIIsafetycabinet.
5. Wipetheoutsideoftheampulewithatissuemoistened(notexcessively)with70%alcoholholdtissueoverampuletoloosenlid.
6. Slowly,dropwise,pipettecellsintopre-warmedgrowthmediumtodiluteouttheDMSO(Prod.No.D2650)(flaskspreparedinStep2).
7. IncubateattheappropriatetemperatureforspeciesandappropriateconcentrationofCO₂inatmosphere.
8. Examinecellsmicroscopically(phasecontrast)after24hoursandsub-cultureasnecessary.

KeyPoints

1. Mosttextbooksrecommendwashingthethawedcellsinmediatoremovethecryoprotectant.Thisisonlynecessaryifthecryoprotectantisknowntohaveanadverseeffectonthecells.Insuchcasesthecellsshouldbewashedinmediabeforebeingaddedtotheirfinalcultureflasks.SeeProtocol7forfurtherdetails.
2. Donotuseanincubatortothawcellculturessincetherateofthawingachievedistooslowresultinginalossofviability.
3. IfaCO₂incubatorisnotavailablegastheflasksfor1-2minuteswith5%CO₂in95%airfilteredthrougha0.25mfilter.
4. Forsomeculturesitisnecessarytosubculturebeforeconfluenceisreachedinordertomaintaintheircharacteristicse.g.thecontactinhibitionofNIH3T3(Prod.No.93061524)cellsislostiftheyareallowedtoreachconfluencerepeatedly.

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