293T說(shuō)明與培養(yǎng)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)來(lái)源293T細(xì)胞系，最初引于

7、293tsA1609neo,是一種可高度轉(zhuǎn)染的衍生于人胚腎293細(xì)胞的細(xì)胞系，包含SV40T抗原。備注該細(xì)胞系可以復(fù)制包含SV40復(fù)制區(qū)域的質(zhì)粒。用于產(chǎn)生逆病毒時(shí)可獲得高滴度。該細(xì)胞系廣泛用于逆病毒生產(chǎn)，基因表達(dá)和蛋白質(zhì)生產(chǎn)。培養(yǎng)方法完全培養(yǎng)基此細(xì)胞系的基礎(chǔ)培養(yǎng)基為ATCC配方的DMEM（貨號(hào)30-2002）。配制完全生長(zhǎng)培養(yǎng)基，在基礎(chǔ)培養(yǎng)基中加入以下成分：10%的胎牛血清（熱滅活）（ATCC30-2020），2mML-谷氨酰胺（ATCC30-2214），1%的青霉素/鏈霉素。傳代體積設(shè)定為75cm2燒瓶。如果是其他型號(hào)，請(qǐng)按體積增加或減少培養(yǎng)液。1. 除去并丟棄培養(yǎng)液。2. 用Ca2+/Mg2+free的（D-PBS）或者0.05%（w/v）胰蛋白酶-0.53mMEDTA溶液來(lái)去除所有包含胰蛋白酶抑制劑的血清殘留。3. 加1.0到2.0mL的胰蛋白酶-EDTA溶液到培養(yǎng)瓶，在倒置顯微鏡下觀察細(xì)胞直到細(xì)胞層脫離（通常在515分鐘）。注：為了避免聚集，在等待細(xì)胞脫落時(shí)不要通過(guò)拍打或者搖動(dòng)培養(yǎng)瓶來(lái)晃動(dòng)細(xì)胞。如果細(xì)胞難以脫離，可以放在37C加速脫離。4. 加入68mL的完全