RPMI Medium 1640（含双抗） 培养基

RPMI Medium 1640由GIBCO干粉配制，过滤灭菌。含双抗、HEPES。保存期长，缓冲能力强。
细胞培养基既是培养细胞中供给细胞营养和促使细胞生殖增殖的基础物质，也是培养细胞生长和繁殖的生存环境。
索莱宝公司生产的培养基精选进口干粉，用超纯去离子水配制，0.1um过滤而成，含双抗，含Hepes。

RPMI Medium 1640（含双抗） 培养基

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。

细胞复苏的注意事项：

1.细胞复苏时要注意融化冻存细胞的应注意融化冻存细胞速度要快，可不时摇动安瓿或冷冻管，使之尽快通过易受损的温度段(-5～0℃)。

2.冻存液对细胞有毒性，解冻后必须用Dhanks洗两遍才能移入培养瓶中培养。培养液中血清不能太少。

3.从液氮拿出来，以快速度丢入37度水浴，等细胞液刚刚化完取出，加培养液稀释。这样可以避免复苏过程中细胞液中有冰晶的出现，冰晶会破坏细胞膜及细胞内部结构的。

4.刚复苏的细胞还是少折腾它好，细胞37°快速解冻后直接放入预热的培养基。

5.DMSO在4度以下对细胞无毒，4度以上有毒；复苏必须尽快除去。要记得慢冻速溶！

6.取细胞的过程中注意带好防冻手套，护目镜。细胞冻存管可能漏入液氮，解冻时冻存管中的气温急剧上升，可导致爆炸。

7.常温下二甲基亚砜（DMSO）对细胞的毒副作用较大，因此，必须在1-2 min内使冻存液*融化。如果复苏温度太低，会造成细胞的损伤，所以选择40℃复苏。8.贴壁细胞复苏实验标准流程是将解冻后的细胞悬液行离心，以去除冷冻保护液DMSO对细胞的损伤，但是离心也会对刚复苏的状态欠佳的细胞产生损伤。也有的实验操作是将解冻后的细胞悬液先直接吹打均匀后分装到培养瓶中进行培养，第二天换液。这可能使培养基中少量的DMSO对细胞造成损伤。

细胞复苏后贴壁细胞较少的原意有哪些：

1.冻存细胞的时候是不是消化时间过长，这是一般人注意不到的地方，要知道太长的消化时间会使细胞复苏时失去贴壁能力。

2.有可能是细胞冻存液的质量，细胞冻存液的质量不好也会导致细胞死亡。

3.冻存液的量加的是不是太多，ATCC推荐是不超过7%，大于5%，太多也不好。

4.冻存的时候是不是把DMSO混均匀，这个有一些影响，但不算太大。

5.冻存时温度梯度是不是把握严格，很多人容易忘却这个事情，因为这个东西流程长。

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