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RPMI Medium 1640（含雙抗） 培養基

RPMI Medium 1640由(you)GIBCO干粉配制(zhi)，過(guo)濾滅菌。含雙抗、HEPES。保存(cun)期長，緩沖能(neng)力強。
細胞(bao)(bao)培(pei)養基(ji)既(ji)是(shi)培(pei)養細胞(bao)(bao)中供給細胞(bao)(bao)營養和(he)促使細胞(bao)(bao)生(sheng)殖增殖的基(ji)礎物質，也是(shi)培(pei)養細胞(bao)(bao)生(sheng)長和(he)繁(fan)殖的生(sheng)存(cun)環境。
索萊寶公司生產的培養基(ji)精選進口干粉(fen)，用超純去離子水配制(zhi)，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(de)(de)細胞，有(you)需(xu)要冷凍保存。冷凍的(de)(de)過程(cheng)稱為凍存，把凍上的(de)(de)細胞化開的(de)(de)過程(cheng)叫(jiao)細胞復(fu)(fu)蘇(su)。細胞復(fu)(fu)蘇(su)是一簡單的(de)(de)試驗操(cao)作，但操(cao)作中有(you)許多需(xu)要注意(yi)的(de)(de)事項，在實驗操(cao)作中注意(yi)細小(xiao)問題，才(cai)能使復(fu)(fu)蘇(su)后的(de)(de)細胞狀態保持良好。

細胞復蘇實驗準備：

主要器材(cai)：一次性(xing)滴管(guan)、打(da)火機、酒精(jing)燈(deng)、大鑷子、中鑷子、記號筆(bi)、廢液(ye)缸、封口膜、剪子。

主(zhu)要試(shi)劑(ji)： PBS、培養液、消化酶(mei)（胰酶(mei)）、75%酒精(jing)、雙(shuang)抗(kang)。

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

三蒸水： 500mL，溶解(jie)后高壓滅菌(jun)。

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(pei)戴眼鏡(jing)和手套，從液氮罐中取出安瓿或冷凍(dong)管。

2.迅速放入38℃水(shui)浴中，并不(bu)時搖動，在(zai)1分鐘內使其*融化，然后在(zai)無菌下取出細胞。

3.在1000r/min速度(du)(du)下離(li)心5～10分鐘，棄去上層液，加(jia)入(ru)適量培(pei)養(yang)(yang)(yang)液后(hou)接(jie)種于培(pei)養(yang)(yang)(yang)瓶中，接(jie)種濃度(du)(du)1×109/L，置37℃溫箱靜置培(pei)養(yang)(yang)(yang)，次日更(geng)換一次培(pei)養(yang)(yang)(yang)液，繼(ji)續(xu)培(pei)養(yang)(yang)(yang)，觀察(cha)生長(chang)情況。若細胞(bao)密度(du)(du)較高(gao)，及時傳代。或(huo)無(wu)需離(li)心直接(jie)將細胞(bao)加(jia)入(ru)瓶中，并(bing)加(jia)入(ru)培(pei)養(yang)(yang)(yang)基貼(tie)壁培(pei)養(yang)(yang)(yang)12～24小時后(hou)，充(chong)去上清，換入(ru)新鮮培(pei)養(yang)(yang)(yang)基繼(ji)續(xu)培(pei)養(yang)(yang)(yang)。

細胞復蘇的注意事項：

1.細胞復蘇(su)時要(yao)(yao)注意(yi)融化凍存(cun)細胞的應注意(yi)融化凍存(cun)細胞速(su)度要(yao)(yao)快，可不時搖動(dong)安瓿(bu)或冷凍管，使之盡快通過易(yi)受(shou)損的溫度段(-5～0℃)。

2.凍存液(ye)對細胞有毒性，解凍后必須(xu)用(yong)Dhanks洗(xi)兩遍才能移入(ru)培(pei)養(yang)瓶中培(pei)養(yang)。培(pei)養(yang)液(ye)中血清不(bu)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(gang)(gang)剛(gang)(gang)化完取出，加培養液稀釋。這樣可以避免復(fu)蘇過程中(zhong)細胞液中(zhong)有冰(bing)晶的出現(xian)，冰(bing)晶會破(po)壞(huai)細胞膜及細胞內部結(jie)構的。

4.剛(gang)復蘇的細胞(bao)還是少折騰(teng)它好，細胞(bao)37°快速(su)解(jie)凍后(hou)直接放入預(yu)熱的培養基。

5.DMSO在4度以(yi)下對細胞(bao)無(wu)毒，4度以(yi)上有毒；復蘇必須盡快除去。要記得慢凍(dong)速溶！

6.取細胞的過程中注(zhu)意(yi)帶好防凍手套，護(hu)目鏡。細胞凍存管可(ke)能漏入(ru)液氮(dan)，解(jie)凍時凍存管中的氣溫(wen)急劇(ju)上升，可(ke)導致爆炸。

7.常溫下二甲基亞砜（DMSO）對細(xi)(xi)胞(bao)的毒副作(zuo)用較大，因此(ci)，必(bi)須在1-2 min內使(shi)凍(dong)存液(ye)*融(rong)化。如果復蘇(su)溫度(du)太低，會造(zao)成細(xi)(xi)胞(bao)的損傷，所以選擇(ze)40℃復蘇(su)。8.貼壁細(xi)(xi)胞(bao)復蘇(su)實驗標準流程是(shi)(shi)將(jiang)解(jie)凍(dong)后(hou)的細(xi)(xi)胞(bao)懸液(ye)行(xing)離心，以去除冷凍(dong)保護液(ye)DMSO對細(xi)(xi)胞(bao)的損傷，但是(shi)(shi)離心也會對剛(gang)復蘇(su)的狀態欠(qian)佳的細(xi)(xi)胞(bao)產(chan)生(sheng)損傷。也有的實驗操作(zuo)是(shi)(shi)將(jiang)解(jie)凍(dong)后(hou)的細(xi)(xi)胞(bao)懸液(ye)先直接吹打均勻后(hou)分裝(zhuang)到培(pei)養瓶中(zhong)進(jin)行(xing)培(pei)養，第二天換液(ye)。這(zhe)可能使(shi)培(pei)養基中(zhong)少量的DMSO對細(xi)(xi)胞(bao)造(zao)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(bao)的時候(hou)是不(bu)是消(xiao)化(hua)時間過長(chang)，這是一般人注(zhu)意不(bu)到的地方，要知(zhi)道太長(chang)的消(xiao)化(hua)時間會使細胞(bao)復蘇(su)時失去貼(tie)壁(bi)能力。

2.有可能是細(xi)胞凍存液(ye)的質(zhi)(zhi)量，細(xi)胞凍存液(ye)的質(zhi)(zhi)量不(bu)好也會導致細(xi)胞死亡。

3.凍存液的量(liang)加的是不(bu)是太多，ATCC推薦是不(bu)超(chao)過7%，大于5%，太多也不(bu)好。

4.凍存的時候是(shi)(shi)不是(shi)(shi)把DMSO混均勻，這個有一些(xie)影響，但不算太大。

5.凍存時溫度(du)梯度(du)是不是把握(wo)嚴格，很(hen)多人容易忘卻這個事(shi)情，因為這個東西流程長。

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