细胞培养 RPMI Medium 1640

平时研究用的细胞，有需要冷冻保存。冷冻的过程称为冻存，把冻上的细胞化开的过程叫细胞复苏。细胞复苏是一简单的试验操作，但操作中有许多需要注意的事项，在实验操作中注意细小问题，才能使复苏后的细胞状态保持良好。

细胞复苏实验准备：

主要器材：一次性滴管、打火机、酒精灯、大镊子、中镊子、记号笔、废液缸、封口膜、剪子。

主要试剂： PBS、培养液、消化酶（胰酶）、75%酒精、双抗。

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

三蒸水： 500mL，溶解后高压灭菌。

培养基配制：5mL双抗、50mL血清，加入培养基中。

细胞复苏操作步骤：

1.佩戴眼镜和手套，从液氮罐中取出安瓿或冷冻管。

2.迅速放入38℃水浴中，并不时摇动，在1分钟内使其*融化，然后在无菌下取出细胞。

3.在1000r/min速度下离心5～10分钟，弃去上层液，加入适量培养液后接种于培养瓶中，接种浓度1×109/L，置37℃温箱静置培养，次日更换一次培养液，继续培养，观察生长情况。若细胞密度较高，及时传代。或无需离心直接将细胞加入瓶中，并加入培养基贴壁培养12～24小时后，充去上清，换入新鲜培养基继续培养。

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细胞培养 RPMI Medium 1640