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RPMI Medium 1640（含雙抗） 培養基

RPMI Medium 1640由GIBCO干粉配制，過濾滅菌。含雙抗、HEPES。保存期長，緩沖能力強。
細胞培養基既是培養細胞中供給細胞營養和促使細胞生殖增殖的基礎物質，也是培養細胞生長和繁殖的生存環境。
索萊寶公司生產的培養基精選進口干粉，用超純去離子水配制，0.1um過濾而成，含雙抗，含Hepes。

RPMI Medium 1640（含雙抗） 培養基

平時研究用的細胞，有需要冷凍保存。冷凍的過程稱為凍存，把凍上的細胞化開的過程叫細胞復蘇。細胞復蘇是一簡單的試驗操作，但操作中有許多需要注意的事項，在實驗操作中注意細小問題，才能使復蘇后的細胞狀態保持良好。

細胞復蘇實驗準備：

主要器材：一次性滴管、打火機、酒精燈、大鑷子、中鑷子、記號筆、廢液缸、封口膜、剪子。

主要試劑： PBS、培養液、消化酶（胰酶）、75%酒精、雙抗。

PBS配制：NaCL :4g   KCL:0.1g   Na2HPO4 :1.745g   KH2PO4 :0.1g

三蒸水： 500mL，溶解后高壓滅菌。

培養基配制：5mL雙抗、50mL血清，加入培養基中。

細胞復蘇操作步驟：

1.佩戴眼鏡和手套，從液氮罐中取出安瓿或冷凍管。

2.迅速放入38℃水浴中，并不時搖動，在1分鐘內使其*融化，然后在無菌下取出細胞。

3.在1000r/min速度下離心5～10分鐘，棄去上層液，加入適量培養液后接種于培養瓶中，接種濃度1×109/L，置37℃溫箱靜置培養，次日更換一次培養液，繼續培養，觀察生長情況。若細胞密度較高，及時傳代。或無需離心直接將細胞加入瓶中，并加入培養基貼壁培養12～24小時后，充去上清，換入新鮮培養基繼續培養。

細胞復蘇的注意事項：

1.細胞復蘇時要注意融化凍存細胞的應注意融化凍存細胞速度要快，可不時搖動安瓿或冷凍管，使之盡快通過易受損的溫度段(-5～0℃)。

2.凍存液對細胞有毒性，解凍后必須用Dhanks洗兩遍才能移入培養瓶中培養。培養液中血清不能太少。

3.從液氮拿出來，以快速度丟入37度水浴，等細胞液剛剛化完取出，加培養液稀釋。這樣可以避免復蘇過程中細胞液中有冰晶的出現，冰晶會破壞細胞膜及細胞內部結構的。

4.剛復蘇的細胞還是少折騰它好，細胞37°快速解凍后直接放入預熱的培養基。

5.DMSO在4度以下對細胞無毒，4度以上有毒；復蘇必須盡快除去。要記得慢凍速溶！

6.取細胞的過程中注意帶好防凍手套，護目鏡。細胞凍存管可能漏入液氮，解凍時凍存管中的氣溫急劇上升，可導致爆炸。

7.常溫下二甲基亞砜（DMSO）對細胞的毒副作用較大，因此，必須在1-2 min內使凍存液*融化。如果復蘇溫度太低，會造成細胞的損傷，所以選擇40℃復蘇。8.貼壁細胞復蘇實驗標準流程是將解凍后的細胞懸液行離心，以去除冷凍保護液DMSO對細胞的損傷，但是離心也會對剛復蘇的狀態欠佳的細胞產生損傷。也有的實驗操作是將解凍后的細胞懸液先直接吹打均勻后分裝到培養瓶中進行培養，第二天換液。這可能使培養基中少量的DMSO對細胞造成損傷。

細胞復蘇后貼壁細胞較少的原意有哪些：

1.凍存細胞的時候是不是消化時間過長，這是一般人注意不到的地方，要知道太長的消化時間會使細胞復蘇時失去貼壁能力。

2.有可能是細胞凍存液的質量，細胞凍存液的質量不好也會導致細胞死亡。

3.凍存液的量加的是不是太多，ATCC推薦是不超過7%，大于5%，太多也不好。

4.凍存的時候是不是把DMSO混均勻，這個有一些影響，但不算太大。

5.凍存時溫度梯度是不是把握嚴格，很多人容易忘卻這個事情，因為這個東西流程長。

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