表达SV40T抗原人胚肾上皮细胞 293FT本公司代理美国ATCC，理德DSMZ，ScienCell德国细胞库，ECACC,ICLC,DSMZ的细胞株以及菌株等品牌细胞。齐一生物大量库存ATCC 细胞，癌细胞，正常细胞，*，质量保证，公司秉承“诚信、专业、高效”的经营理念为广大客户服务。欢迎各位老师！齐一生物科技(上海）有限公司：

表达SV40T抗原人胚肾上皮细胞 293FT细胞培养方法 
1、收到细胞，请查看瓶子是否有破裂，培养基是否漏出，是否浑浊，如有请尽快。
2、收到细胞，如包装完好，请在显微镜下观察细胞。,由于运输过程中的问题，细胞培养瓶中的贴壁细胞有可能从瓶壁中脱落下来，显微镜下观察会出现细胞悬浮的情况，出现此状态时，请不要打开细胞培养瓶，应立即将培养瓶置于细胞培养箱里静止3-5小时左右，让细胞先稳定下，再于显微镜下观察，此时多数细胞会重新贴附于瓶壁。如细胞仍不能贴壁，请用台盼蓝染色法鉴定细胞活力，如台盼蓝染色证实细胞活力正常请按悬浮细胞的方法处理
3. 收到细胞时如无异常情况 ，请在显微镜下观察细胞密度，如为贴壁细胞，未超过80%汇合度时，将培养瓶中培养基吸出，留下 5-10ML培养基继续培养：超过80%汇合度时，请按细胞培养条件传代培养。如为悬浮细胞，吸出培养液，1000转/分钟离心3分钟，吸出上清，管底细胞用新鲜培养基悬浮细胞后移回培养瓶。
4，将培养瓶置于37℃培养箱中培养，盖子微微拧松。吸出的培养基可以保存在灭菌过的瓶子里，存放于4℃冰箱，以备不时之需。
5 、24小时后，细胞形态已恢复并贴满瓶壁，就可以传代了。（贴壁细胞）将培养瓶里的培养基倒去，加3-5ml（以能覆盖细胞生长面为准）PBS或Hanks’液洗涤后弃去。加0.5-1ml  0.25%含EDTA的胰酶消化，消化时间以具体细胞为准，一般1-3分钟，不超过5分钟。可以放入37℃培养箱消化。轻轻晃动瓶壁，见细胞脱落下来，加入3-5ml培养基终止消化。用移液管轻轻吹打瓶壁上的细胞，使之*脱落，然后将溶液吸入离心管内离心，1000rpm/5min。弃上清，视细胞数量决定分瓶数，一般一传二，如细胞量多可一传三，有些细胞不易传得过稀，有些生长较快的细胞则可以多传几瓶，以具体细胞和经验为准。（悬浮细胞）用移液管轻轻吹打瓶壁，直接将溶液吸入离心管离心即可。
6，贴壁细胞 ，悬浮细胞。严格无菌操作。换液时，换新的细胞培养瓶和换新鲜的培养液，37度    5%CO2 培养
7.  收到细胞后，请镜下观察细胞，用恰当方式处理细胞。若悬浮的细胞较多，请离心收集细胞，接种到一个新的培养瓶中。弃掉原液，使用新鲜配制的培养基，使用进口胎牛血清. 刚接到细胞，若细胞不多时 血清浓度可以加到15％去培养。若细胞迏到80%左右 ，血清浓度还是在10％。
培养基参考
500ml基础培养基（含：必需与非必需氨基酸、维他命、有机与无机成分、激素、生长因子与微量元素）、细胞生长添加剂、抗生素/抗菌素溶液或青霉素/链霉素（P/S）溶液、FBS，适合在饱和湿度为5%CO2与95%空气的培养箱中使用

一、细胞操作参考书
二、生长特性：贴壁    悬浮
三、细胞生长条件：
培养基                  90%              （GIBCO）
血清              10%  FBS（GIBCO）
温度                       37℃
空气条件      5% CO2，,95% AIR
生长代数    P4
冻存条件    培养基70%、血清20%、DMSO10%
四、组成：
组份    规格
细胞一瓶    T25
细胞培养与操作说明    1份
五、细胞接收后的操作流程与注意事项
1.如果细胞为贴壁细胞，，而收到时呈悬浮或者部分悬浮的状态，请将悬浮的细胞即时离心，加15%血清的*培养基到新的培养皿/瓶继续培养3天；同时原培养瓶中剩下的贴壁细胞更换为15%血清的*培养基，培养3天。3天后若原瓶或者新瓶中的细胞都没有出现增殖而是继续脱落死亡，请及时实验室，技术人员会跟进解决
2.贴壁细胞生长缓慢：适当提高血清浓度（zui高不能超过20%），或可根据该细胞生长密度，考虑胰酶消化后，转移到新的培养瓶继续培养
3.生长不均：贴壁细胞若出现分布不均，成岛状生长，可将细胞进行消化，重悬打散细胞，加入新鲜培养基进行培养

六、细胞常规培养传代流程（请严格遵照无菌操作）
1．吸出原培养瓶中的培养基，PBS缓冲液润洗细胞两次，加2~3 ml  0.25%胰酶进行消化细胞（注意把握消化时间，通常控制在1-2min）
2．镜下观察消化情况，在细胞边缘缩小，贴壁松动时（不建议消化到细胞漂浮）去掉胰酶，加6~8ml *培养基，轻轻吹打细胞层，尽量把细胞层吹落，吹散
3．取部分细胞悬液转移到新的培养皿/瓶中，添加适当的*培养基，，把细胞悬液打匀，于培养箱中培养
4．注意培养基PH值变化情况，定期换液（每周2-3次），待细胞密度达到80%以后重复1项操作或者冻存
特别注意：（如使用公共实验室或初次接触细胞培养，建议添加双抗培养）
1.收到细胞后请尽快更换为含15%血清的新鲜培养基，如因特殊情况需要继续使用原瓶培养基，请在原瓶培养基中额外添加10%的血清（原瓶培养基的继续使用时间zui长不宜超过72小时）
2.贴壁细胞收到当天切忌立刻消化，请将细胞放置培养箱孵育过夜到第二天再做消化传代
如签收时出现培养瓶壁破裂，漏液等情况请及时做好照片记录并实验室
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