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RPMI Medium 1640（含雙抗） 培養基

RPMI Medium 1640由GIBCO干粉配制(zhi)，過(guo)濾滅菌。含雙抗、HEPES。保存期(qi)長，緩沖能力強(qiang)。
細(xi)胞(bao)培(pei)養基(ji)既是培(pei)養細(xi)胞(bao)中供(gong)給細(xi)胞(bao)營養和促(cu)使細(xi)胞(bao)生殖增(zeng)殖的基(ji)礎物(wu)質(zhi)，也(ye)是培(pei)養細(xi)胞(bao)生長和繁(fan)殖的生存環境。
索(suo)萊寶公(gong)司生產的(de)培養基精選(xuan)進口干粉，用超純(chun)去離子水配(pei)制(zhi)，0.1um過濾而(er)成，含雙(shuang)抗，含Hepes。

RPMI Medium 1640（含雙抗） 培養基

平時研(yan)究用的(de)細胞，有需(xu)要冷凍保存。冷凍的(de)過程稱為凍存，把凍上的(de)細胞化(hua)開(kai)的(de)過程叫(jiao)細胞復蘇(su)(su)。細胞復蘇(su)(su)是一簡單(dan)的(de)試驗操作，但(dan)操作中有許多(duo)需(xu)要注意的(de)事項，在(zai)實驗操作中注意細小問題，才能使復蘇(su)(su)后的(de)細胞狀態保持良好。

細胞復蘇實驗準備：

主要器材：一次性滴(di)管、打火機、酒精燈、大鑷子(zi)(zi)、中鑷子(zi)(zi)、記號筆、廢液缸、封口膜、剪(jian)子(zi)(zi)。

主(zhu)要試劑： PBS、培養液、消(xiao)化(hua)酶(mei)（胰酶(mei)）、75%酒精、雙抗。

PBS配(pei)制：NaCL :4g   KCL:0.1g   Na2HPO4 :1.745g   KH2PO4 :0.1g

三(san)蒸水： 500mL，溶解后高壓滅菌。

培養基配制：5mL雙抗、50mL血清(qing)，加入培養基中。

細胞復蘇操作步驟：

1.佩戴(dai)眼鏡和手套，從(cong)液(ye)氮罐中取出安瓿或冷凍管。

2.迅速放入38℃水浴中，并不時搖動，在(zai)1分鐘內使(shi)其*融(rong)化，然后在(zai)無菌(jun)下(xia)取出細胞。

3.在1000r/min速度(du)下離心5～10分鐘，棄去上層液(ye)(ye)，加(jia)入(ru)適量培養(yang)(yang)液(ye)(ye)后(hou)接(jie)種于培養(yang)(yang)瓶中，接(jie)種濃度(du)1×109/L，置(zhi)37℃溫(wen)箱靜置(zhi)培養(yang)(yang)，次日更換一次培養(yang)(yang)液(ye)(ye)，繼(ji)續培養(yang)(yang)，觀察(cha)生長情(qing)況。若細(xi)胞密度(du)較(jiao)高，及時傳(chuan)代。或(huo)無需離心直接(jie)將細(xi)胞加(jia)入(ru)瓶中，并(bing)加(jia)入(ru)培養(yang)(yang)基貼壁培養(yang)(yang)12～24小時后(hou)，充去上清，換入(ru)新鮮培養(yang)(yang)基繼(ji)續培養(yang)(yang)。

細胞復蘇的注意事項：

1.細(xi)胞復蘇時(shi)要(yao)注意(yi)融化凍(dong)存細(xi)胞的(de)應注意(yi)融化凍(dong)存細(xi)胞速度要(yao)快(kuai)，可(ke)不時(shi)搖(yao)動安瓿或冷凍(dong)管(guan)，使之盡快(kuai)通過易(yi)受損(sun)的(de)溫度段(-5～0℃)。

2.凍存液(ye)對細胞有(you)毒性(xing)，解(jie)凍后(hou)必須用Dhanks洗(xi)兩遍才能移入培養(yang)瓶中培養(yang)。培養(yang)液(ye)中血清不能太少。

3.從液(ye)(ye)氮(dan)拿出(chu)來，以快速度丟入37度水浴，等細(xi)胞液(ye)(ye)剛剛化完取出(chu)，加培養液(ye)(ye)稀釋。這(zhe)樣可(ke)以避免(mian)復蘇過程中細(xi)胞液(ye)(ye)中有冰晶的(de)出(chu)現(xian)，冰晶會(hui)破(po)壞細(xi)胞膜及細(xi)胞內部(bu)結(jie)構的(de)。

4.剛(gang)復蘇的(de)細胞還是少折騰(teng)它好，細胞37°快速解凍后直(zhi)接(jie)放入預(yu)熱的(de)培養基。

5.DMSO在4度(du)以下對細胞無毒，4度(du)以上有毒；復蘇必須(xu)盡快除去。要記得慢凍速(su)溶！

6.取細胞的過程中注意帶好防凍(dong)手(shou)套，護(hu)目鏡。細胞凍(dong)存(cun)管(guan)可能漏(lou)入(ru)液氮，解(jie)凍(dong)時(shi)凍(dong)存(cun)管(guan)中的氣(qi)溫(wen)急劇上升，可導致爆(bao)炸。

7.常溫下二甲基亞砜（DMSO）對(dui)細(xi)(xi)胞(bao)(bao)(bao)的(de)(de)毒副作(zuo)用(yong)較(jiao)大，因此，必(bi)須在1-2 min內使凍存(cun)液(ye)(ye)*融(rong)化。如果(guo)復蘇溫度太低，會(hui)造成細(xi)(xi)胞(bao)(bao)(bao)的(de)(de)損(sun)(sun)(sun)傷，所(suo)以選擇40℃復蘇。8.貼壁(bi)細(xi)(xi)胞(bao)(bao)(bao)復蘇實驗(yan)標(biao)準流程是將(jiang)解(jie)凍后的(de)(de)細(xi)(xi)胞(bao)(bao)(bao)懸液(ye)(ye)行離(li)心(xin)，以去除冷凍保(bao)護液(ye)(ye)DMSO對(dui)細(xi)(xi)胞(bao)(bao)(bao)的(de)(de)損(sun)(sun)(sun)傷，但是離(li)心(xin)也(ye)會(hui)對(dui)剛復蘇的(de)(de)狀態欠佳的(de)(de)細(xi)(xi)胞(bao)(bao)(bao)產生損(sun)(sun)(sun)傷。也(ye)有的(de)(de)實驗(yan)操作(zuo)是將(jiang)解(jie)凍后的(de)(de)細(xi)(xi)胞(bao)(bao)(bao)懸液(ye)(ye)先直(zhi)接(jie)吹打(da)均勻(yun)后分裝到培(pei)養(yang)瓶(ping)中進行培(pei)養(yang)，第二天換液(ye)(ye)。這可能使培(pei)養(yang)基中少量的(de)(de)DMSO對(dui)細(xi)(xi)胞(bao)(bao)(bao)造成損(sun)(sun)(sun)傷。

細胞復蘇后貼壁細胞較少的原意有哪些：

1.凍存(cun)細胞的時(shi)候是(shi)不是(shi)消化時(shi)間過長，這(zhe)是(shi)一(yi)般人注意不到的地方，要知道太(tai)長的消化時(shi)間會使細胞復蘇時(shi)失(shi)去貼壁能力。

2.有可能是細胞凍存液的質量，細胞凍存液的質量不好也會(hui)導致細胞死亡。

3.凍存液的(de)量加的(de)是不(bu)是太多(duo)，ATCC推薦是不(bu)超過(guo)7%，大(da)于5%，太多(duo)也不(bu)好。

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

5.凍存時溫度梯度是(shi)不是(shi)把握嚴格(ge)，很多人容易(yi)忘卻這(zhe)(zhe)個事情，因為這(zhe)(zhe)個東西流(liu)程長。

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