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RPMI Medium 1640（含雙抗） 培養(yǎng)基

RPMI Medium 1640由GIBCO干粉配制，過(guò)濾滅菌。含雙抗、HEPES。保存期長(zhǎng)，緩沖能力強(qiáng)。
細(xì)胞培養(yǎng)基既是培養(yǎng)細(xì)胞中供給細(xì)胞營(yíng)養(yǎng)和促使細(xì)胞生殖增殖的基礎(chǔ)物質(zhì)，也是培養(yǎng)細(xì)胞生長(zhǎng)和繁殖的生存環(huán)境。
索萊寶公司生產(chǎn)的培養(yǎng)基精選進(jìn)口干粉，用超純?nèi)ルx子水配制，0.1um過(guò)濾而成，含雙抗，含Hepes。

RPMI Medium 1640（含雙抗） 培養(yǎng)基

平時(shí)研究用的細(xì)胞，有需要冷凍保存。冷凍的過(guò)程稱為凍存，把凍上的細(xì)胞化開(kāi)的過(guò)程叫細(xì)胞復(fù)蘇。細(xì)胞復(fù)蘇是一簡(jiǎn)單的試驗(yàn)操作，但操作中有許多需要注意的事項(xiàng)，在實(shí)驗(yàn)操作中注意細(xì)小問(wèn)題，才能使復(fù)蘇后的細(xì)胞狀態(tài)保持良好。

細(xì)胞復(fù)蘇實(shí)驗(yàn)準(zhǔn)備：

主要器材：一次性滴管、打火機(jī)、酒精燈、大鑷子、中鑷子、記號(hào)筆、廢液缸、封口膜、剪子。

主要試劑： PBS、培養(yǎng)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(yǎng)基配制：5mL雙抗、50mL血清，加入培養(yǎng)基中。

細(xì)胞復(fù)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(shí)搖動(dòng)，在1分鐘內(nèi)使其*融化，然后在無(wú)菌下取出細(xì)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(yǎng)液后接種于培養(yǎng)瓶中，接種濃度1×109/L，置37℃溫箱靜置培養(yǎng)，次日更換一次培養(yǎng)液，繼續(xù)培養(yǎng)，觀察生長(zhǎng)情況。若細(xì)胞密度較高，及時(shí)傳代?；驘o(wú)需離心直接將細(xì)胞加入瓶中，并加入培養(yǎng)基貼壁培養(yǎng)12～24小時(shí)后，充去上清，換入新鮮培養(yǎng)基繼續(xù)培養(yǎng)。

細(xì)胞復(fù)蘇的注意事項(xiàng)：

1.細(xì)胞復(fù)蘇時(shí)要注意融化凍存細(xì)胞的應(yīng)注意融化凍存細(xì)胞速度要快，可不時(shí)搖動(dòng)安瓿或冷凍管，使之盡快通過(guò)易受損的溫度段(-5～0℃)。

2.凍存液對(duì)細(xì)胞有毒性，解凍后必須用Dhanks洗兩遍才能移入培養(yǎng)瓶中培養(yǎng)。培養(yǎng)液中血清不能太少。

3.從液氮拿出來(lái)，以快速度丟入37度水浴，等細(xì)胞液剛剛化完取出，加培養(yǎng)液稀釋。這樣可以避免復(fù)蘇過(guò)程中細(xì)胞液中有冰晶的出現(xiàn)，冰晶會(huì)破壞細(xì)胞膜及細(xì)胞內(nèi)部結(jié)構(gòu)的。

4.剛復(fù)蘇的細(xì)胞還是少折騰它好，細(xì)胞37°快速解凍后直接放入預(yù)熱的培養(yǎng)基。

5.DMSO在4度以下對(duì)細(xì)胞無(wú)毒，4度以上有毒；復(fù)蘇必須盡快除去。要記得慢凍速溶！

6.取細(xì)胞的過(guò)程中注意帶好防凍手套，護(hù)目鏡。細(xì)胞凍存管可能漏入液氮，解凍時(shí)凍存管中的氣溫急劇上升，可導(dǎo)致爆炸。

7.常溫下二甲基亞砜（DMSO）對(duì)細(xì)胞的毒副作用較大，因此，必須在1-2 min內(nèi)使凍存液*融化。如果復(fù)蘇溫度太低，會(huì)造成細(xì)胞的損傷，所以選擇40℃復(fù)蘇。8.貼壁細(xì)胞復(fù)蘇實(shí)驗(yàn)標(biāo)準(zhǔn)流程是將解凍后的細(xì)胞懸液行離心，以去除冷凍保護(hù)液DMSO對(duì)細(xì)胞的損傷，但是離心也會(huì)對(duì)剛復(fù)蘇的狀態(tài)欠佳的細(xì)胞產(chǎn)生損傷。也有的實(shí)驗(yàn)操作是將解凍后的細(xì)胞懸液先直接吹打均勻后分裝到培養(yǎng)瓶中進(jìn)行培養(yǎng)，第二天換液。這可能使培養(yǎng)基中少量的DMSO對(duì)細(xì)胞造成損傷。

細(xì)胞復(fù)蘇后貼壁細(xì)胞較少的原意有哪些：

1.凍存細(xì)胞的時(shí)候是不是消化時(shí)間過(guò)長(zhǎng)，這是一般人注意不到的地方，要知道太長(zhǎng)的消化時(shí)間會(huì)使細(xì)胞復(fù)蘇時(shí)失去貼壁能力。

2.有可能是細(xì)胞凍存液的質(zhì)量，細(xì)胞凍存液的質(zhì)量不好也會(huì)導(dǎo)致細(xì)胞死亡。

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

4.凍存的時(shí)候是不是把DMSO混均勻，這個(gè)有一些影響，但不算太大。

5.凍存時(shí)溫度梯度是不是把握嚴(yán)格，很多人容易忘卻這個(gè)事情，因?yàn)檫@個(gè)東西流程長(zhǎng)。

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