293T说明与培养方法

1、293T(ATCCCRL-321)DerivationThe293Tcellline,originallyreferredas293tsA1609neo,isahighlytransfectablederivativeofhumanembryonickidney293cells,andcontainstheSV40T-antigen.CommentsThiscelllineiscompetenttoreplicatevectorscarryingtheSV40regionofreplication.Itgiveshightiterswhenusedtoproduceretroviruses.I

2、thasbeenwidelyusedforretroviralproduction,geneexpressionandproteinproduction.CultureMethodCompleteGrowthMediumThebasemediumforthiscelllineisDulbecco'sModifiedEagle'sMedium(DMEM)(ATCC30-2002).Tomakethecompletegrowthmedium,addthefollowingcomponentstothebasemedium:10%FetalBovineSerum(heatinacti

3、vated)(ATCC30-2020),2mML-glutamine(ATCC30-2214),1%Penicillin/Streptomycin.SubculturingVolumesusedinthisprotocolarefor75cm2flasks;proportionallyreduceorincreaseamountofdissociationmediumforculturevesselsofothersizes.1. Removeanddiscardculturemedium.2. BrieflyrinsethecelllayerwithCa+/Mg+freeDulbecco&#

4、39;sphosphate-bufferedsaline(D-PBS)or0.05%(w/v)Trypsin-0.53mMEDTAsolutiontoremovealltracesofserumwhichcontainstrypsininhibitor.3. Add1.0to2.0mLofTrypsin-EDTAsolutiontoflaskandobservecellsunderaninvertedmicroscopeuntilcelllayerisdispersed(usuallywithin5to15minutes).Note:Toavoidclumpingdonotagitatethe

5、cellsbyhittingorshakingtheflaskwhilewaitingforthecellstodetach.Cellsthataredifficulttodetachmaybeplacedat37°Ctofacilitatedispersal.4. Add6.0to8.0mLofcompletegrowthmediumandaspiratecellsbygentlypipetting.5. Addappropriatealiquotsofthecellsuspensiontonewculturevessels.Incubateculturesat37°C.

6、Subcultivationratio:Asubcultivationratioof1:3to1:8isrecommended.Mediumrenewal:Every2to3daysCryopreservationFreezemedium:completegrowthmediumsupplementedwith5%(v/v)DMSOStoragetemperature:liquidnitrogenvaporphaseTemperature:37°CAtmosphere:air,95%;carbondioxide(CO2)293T(ATCCCRL-3216)来源293T细胞系，最初引于

7、293tsA1609neo,是一种可高度转染的衍生于人胚肾293细胞的细胞系，包含SV40T抗原。备注该细胞系可以复制包含SV40复制区域的质粒。用于产生逆病毒时可获得高滴度。该细胞系广泛用于逆病毒生产，基因表达和蛋白质生产。培养方法完全培养基此细胞系的基础培养基为ATCC配方的DMEM（货号30-2002）。配制完全生长培养基，在基础培养基中加入以下成分：10%的胎牛血清（热灭活）（ATCC30-2020），2mML-谷氨酰胺（ATCC30-2214），1%的青霉素/链霉素。传代体积设定为75cm2烧瓶。如果是其他型号，请按体积增加或减少培养液。1. 除去并丢弃培养液。2. 用Ca2+/Mg2+free的（D-PBS）或者0.05%（w/v）胰蛋白酶-0.53mMEDTA溶液来去除所有包含胰蛋白酶抑制剂的血清残留。3. 加1.0到2.0mL的胰蛋白酶-EDTA溶液到培养瓶，在倒置显微镜下观察细胞直到细胞层脱离（通常在515分钟）。注：为了避免聚集，在等待细胞脱落时不要通过拍打或者摇动培养瓶来晃动细胞。如果细胞难以脱离，可以放在37C加速脱离。4. 加入68mL的完全