右图为显微镜下看到的血涂片一个视野请据图回答: ①.当人体发生...
来自 : 蚂蚁淘
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
- ReadTechnicaldatasheettoestablishspecificrequirementsforyourcellline.
- Preparetheflasksasappropriate(informationontechnicaldatasheet).Labelwithcelllinename,passagenumberanddate.
- Collectampuleofcellsfromliquidnitrogenstoragewearingappropriateprotectiveequipmentandtransfertolaboratoryinasealedcontainer.
- Stillwearingprotectiveclothing,removeampulefromcontainerandplaceinawaterbathatanappropriatetemperatureforyourcelllinee.g.37ºCformammaliancells.Submergeonlythelowerhalfoftheampule.Allowtothawuntilasmallamountoficeremainsinthevial-usually1-2minutes.TransfertoclassIIsafetycabinet.
- Wipetheoutsideoftheampulewithatissuemoistened(notexcessively)with70%alcoholholdtissueoverampuletoloosenlid.
- Slowly,dropwise,pipettecellsintopre-warmedgrowthmediumtodiluteouttheDMSO(Prod.No.D2650)(flaskspreparedinStep2).
- IncubateattheappropriatetemperatureforspeciesandappropriateconcentrationofCO2inatmosphere.
- Examinecellsmicroscopically(phasecontrast)after24hoursandsub-cultureasnecessary.
KeyPoints
- Mosttextbooksrecommendwashingthethawedcellsinmediatoremovethecryoprotectant.Thisisonlynecessaryifthecryoprotectantisknowntohaveanadverseeffectonthecells.Insuchcasesthecellsshouldbewashedinmediabeforebeingaddedtotheirfinalcultureflasks.SeeProtocol7forfurtherdetails.
- Donotuseanincubatortothawcellculturessincetherateofthawingachievedistooslowresultinginalossofviability.
- IfaCO2incubatorisnotavailablegastheflasksfor1-2minuteswith5%CO2in95%airfilteredthrougha0.25mfilter.
- Forsomeculturesitisnecessarytosubculturebeforeconfluenceisreachedinordertomaintaintheircharacteristicse.g.thecontactinhibitionofNIH3T3(Prod.No.93061524)cellsislostiftheyareallowedtoreachconfluencerepeatedly.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
