細(xì)胞培養(yǎng) RPMI Medium 1640

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)。

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細(xì)胞培養(yǎng) RPMI Medium 1640