表達SV40T抗原人胚腎上皮細胞 293FT本公司代理美國ATCC，理德DSMZ，ScienCell德國細胞庫，ECACC,ICLC,DSMZ的細胞株以及菌株等品牌細胞。齊一生物大量庫存ATCC 細胞，癌細胞，正常細胞，*，質量保證，公司秉承“誠信、專業、高效"的經營理念為廣大客戶服務。歡迎各位老師！齊一生物科技(上海）有限公司：

表達SV40T抗原人胚腎上皮細胞 293FT細胞培養方法 
1、收到細胞，請查看瓶子是否有破裂，培養基是否漏出，是否渾濁，如有請盡快。
2、收到細胞，如包裝完好，請在顯微鏡下觀察細胞。,由于運輸過程中的問題，細胞培養瓶中的貼壁細胞有可能從瓶壁中脫落下來，顯微鏡下觀察會出現細胞懸浮的情況，出現此狀態時，請不要打開細胞培養瓶，應立即將培養瓶置于細胞培養箱里靜止3-5小時左右，讓細胞先穩定下，再于顯微鏡下觀察，此時多數細胞會重新貼附于瓶壁。如細胞仍不能貼壁，請用臺盼藍染色法鑒定細胞活力，如臺盼藍染色證實細胞活力正常請按懸浮細胞的方法處理
3. 收到細胞時如無異常情況 ，請在顯微鏡下觀察細胞密度，如為貼壁細胞，未超過80%匯合度時，將培養瓶中培養基吸出，留下 5-10ML培養基繼續培養：超過80%匯合度時，請按細胞培養條件傳代培養。如為懸浮細胞，吸出培養液，1000轉/分鐘離心3分鐘，吸出上清，管底細胞用新鮮培養基懸浮細胞后移回培養瓶。
4，將培養瓶置于37℃培養箱中培養，蓋子微微擰松。吸出的培養基可以保存在滅菌過的瓶子里，存放于4℃冰箱，以備不時之需。
5 、24小時后，細胞形態已恢復并貼滿瓶壁，就可以傳代了。（貼壁細胞）將培養瓶里的培養基倒去，加3-5ml（以能覆蓋細胞生長面為準）PBS或Hanks’液洗滌后棄去。加0.5-1ml  0.25%含EDTA的胰酶消化，消化時間以具體細胞為準，一般1-3分鐘，不超過5分鐘。可以放入37℃培養箱消化。輕輕晃動瓶壁，見細胞脫落下來，加入3-5ml培養基終止消化。用移液管輕輕吹打瓶壁上的細胞，使之*脫落，然后將溶液吸入離心管內離心，1000rpm/5min。棄上清，視細胞數量決定分瓶數，一般一傳二，如細胞量多可一傳三，有些細胞不易傳得過稀，有些生長較快的細胞則可以多傳幾瓶，以具體細胞和經驗為準。（懸浮細胞）用移液管輕輕吹打瓶壁，直接將溶液吸入離心管離心即可。
6，貼壁細胞 ，懸浮細胞。嚴格無菌操作。換液時，換新的細胞培養瓶和換新鮮的培養液，37度    5%CO2 培養
7.  收到細胞后，請鏡下觀察細胞，用恰當方式處理細胞。若懸浮的細胞較多，請離心收集細胞，接種到一個新的培養瓶中。棄掉原液，使用新鮮配制的培養基，使用進口胎牛血清. 剛接到細胞，若細胞不多時 血清濃度可以加到15％去培養。若細胞迏到80%左右 ，血清濃度還是在10％。
培養基參考
500ml基礎培養基（含：必需與非必需氨基酸、維他命、有機與無機成分、激素、生長因子與微量元素）、細胞生長添加劑、抗生素/抗菌素溶液或青霉素/鏈霉素（P/S）溶液、FBS，適合在飽和濕度為5%CO2與95%空氣的培養箱中使用

一、細胞操作參考書
二、生長特性：貼壁    懸浮
三、細胞生長條件：
培養基                  90%              （GIBCO）
血清              10%  FBS（GIBCO）
溫度                       37℃
空氣條件      5% CO2，,95% AIR
生長代數    P4
凍存條件    培養基70%、血清20%、DMSO10%
四、組成：
組份    規格
細胞一瓶    T25
細胞培養與操作說明    1份
五、細胞接收后的操作流程與注意事項
1.如果細胞為貼壁細胞，，而收到時呈懸浮或者部分懸浮的狀態，請將懸浮的細胞即時離心，加15%血清的*培養基到新的培養皿/瓶繼續培養3天；同時原培養瓶中剩下的貼壁細胞更換為15%血清的*培養基，培養3天。3天后若原瓶或者新瓶中的細胞都沒有出現增殖而是繼續脫落死亡，請及時實驗室，技術人員會跟進解決
2.貼壁細胞生長緩慢：適當提高血清濃度（zui高不能超過20%），或可根據該細胞生長密度，考慮胰酶消化后，轉移到新的培養瓶繼續培養
3.生長不均：貼壁細胞若出現分布不均，成島狀生長，可將細胞進行消化，重懸打散細胞，加入新鮮培養基進行培養

六、細胞常規培養傳代流程（請嚴格遵照無菌操作）
1．吸出原培養瓶中的培養基，PBS緩沖液潤洗細胞兩次，加2~3 ml  0.25%胰酶進行消化細胞（注意把握消化時間，通常控制在1-2min）
2．鏡下觀察消化情況，在細胞邊緣縮小，貼壁松動時（不建議消化到細胞漂浮）去掉胰酶，加6~8ml *培養基，輕輕吹打細胞層，盡量把細胞層吹落，吹散
3．取部分細胞懸液轉移到新的培養皿/瓶中，添加適當的*培養基，，把細胞懸液打勻，于培養箱中培養
4．注意培養基PH值變化情況，定期換液（每周2-3次），待細胞密度達到80%以后重復1項操作或者凍存
特別注意：（如使用公共實驗室或初次接觸細胞培養，建議添加雙抗培養）
1.收到細胞后請盡快更換為含15%血清的新鮮培養基，如因特殊情況需要繼續使用原瓶培養基，請在原瓶培養基中額外添加10%的血清（原瓶培養基的繼續使用時間zui長不宜超過72小時）
2.貼壁細胞收到當天切忌立刻消化，請將細胞放置培養箱孵育過夜到第二天再做消化傳代
如簽收時出現培養瓶壁破裂，漏液等情況請及時做好照片記錄并實驗室
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