大(Da)鼠(Shu)视(Shi)网(Wang)膜(Mo)Müller细(Xi)胞(Bao)永(Yong)生(Sheng)化(Hua)细(Xi)胞(Bao)操(Cao)作(Zuo)步(Bu)骤(Zhou)
大(Da)鼠(Shu)视(Shi)网(Wang)膜(Mo)Mü(?)ller细(Xi)胞(Bao)永(Yong)生(Sheng)化(Hua)细(Xi)胞(Bao)操(Cao)作(Zuo)步(Bu)骤(Zhou)
** 细(Xi)胞(Bao)转(Zhuan)染(Ran)与(Yu)筛(Shai)选(Xuan)**
完(Wan)成(Cheng)细(Xi)胞(Bao)培(Pei)养(Yang)和(He)预(Yu)处(Chu)理(Li)后(Hou),(?)将(Jiang)构(Gou)建(Jian)好(Hao)的(De)SV40大(Da)T抗(Kang)原(Yuan)表(Biao)达(Da)质(Zhi)粒(Li)通(Tong)过(Guo)脂(Zhi)质(Zhi)体(Ti)转(Zhuan)染(Ran)法(Fa)导(Dao)入(Ru)原(Yuan)代(Dai)Mü(?)ller细(Xi)胞(Bao)。(?)转(Zhuan)染(Ran)48小(Xiao)时(Shi)后(Hou),(?)更(Geng)换(Huan)为(Wei)含(Han)400μ(?)g/mL G418的(De)选(Xuan)择(Ze)培(Pei)养(Yang)基(Ji)进(Jin)行(Xing)抗(Kang)性(Xing)筛(Shai)选(Xuan),(?)持(Chi)续(Xu)2-3周(Zhou)直(Zhi)至(Zhi)未(Wei)转(Zhuan)染(Ran)细(Xi)胞(Bao)死(Si)亡(Wang)。(?)期(Qi)间(Jian)需(Xu)每(Mei)3天(Tian)更(Geng)换(Huan)一(Yi)次(Ci)筛(Shai)选(Xuan)培(Pei)养(Yang)基(Ji),(?)并(Bing)密(Mi)切(Qie)观(Guan)察(Cha)细(Xi)胞(Bao)状(Zhuang)态(Tai)。(?)注(Zhu)意(Yi)设(She)置(Zhi)未(Wei)转(Zhuan)染(Ran)细(Xi)胞(Bao)作(Zuo)为(Wei)阴(Yin)性(Xing)对(Dui)照(Zhao),(?)以(Yi)确(Que)认(Ren)筛(Shai)选(Xuan)效(Xiao)果(Guo)。(?)
**单(Dan)克(Ke)隆(Long)分(Fen)离(Li)与(Yu)扩(Kuo)增(Zeng)**
采(Cai)用(Yong)有(You)限(Xian)稀(Xi)释(Shi)法(Fa)进(Jin)行(Xing)单(Dan)克(Ke)隆(Long)分(Fen)离(Li):(?)将(Jiang)筛(Shai)选(Xuan)后(Hou)的(De)细(Xi)胞(Bao)悬(Xuan)液(Ye)稀(Xi)释(Shi)至(Zhi)0.5个(Ge)细(Xi)胞(Bao)/100μ(?)L,(?)接(Jie)种(Zhong)于(Yu)96孔(Kong)板(Ban),(?)每(Mei)孔(Kong)加(Jia)入(Ru)100μ(?)L培(Pei)养(Yang)基(Ji)。(?)培(Pei)养(Yang)1周(Zhou)后(Hou)标(Biao)记(Ji)单(Dan)个(Ge)细(Xi)胞(Bao)形(Xing)成(Cheng)的(De)克(Ke)隆(Long),(?)待(Dai)克(Ke)隆(Long)长(Chang)至(Zhi)孔(Kong)底(Di)50%面(Mian)积(Ji)时(Shi),(?)用(Yong)克(Ke)隆(Long)环(Huan)法(Fa)分(Fen)离(Li)并(Bing)转(Zhuan)移(Yi)至(Zhi)24孔(Kong)板(Ban)扩(Kuo)大(Da)培(Pei)养(Yang)。(?)此(Ci)过(Guo)程(Cheng)需(Xu)特(Te)别(Bie)注(Zhu)意(Yi)无(Wu)菌(Jun)操(Cao)作(Zuo),(?)建(Jian)议(Yi)每(Mei)个(Ge)克(Ke)隆(Long)保(Bao)留(Liu)3个(Ge)备(Bei)份(Fen)。(?)
**永(Yong)生(Sheng)化(Hua)验(Yan)证(Zheng)**
通(Tong)过(Guo)以(Yi)下(Xia)方(Fang)法(Fa)验(Yan)证(Zheng)细(Xi)胞(Bao)永(Yong)生(Sheng)化(Hua)特(Te)性(Xing):(?)
1) RT-PCR检(Jian)测(Ce)SV40大(Da)T抗(Kang)原(Yuan)mRNA表(Biao)达(Da);(?)
2) Western blot检(Jian)测(Ce)大(Da)T抗(Kang)原(Yuan)蛋(Dan)白(Bai)表(Biao)达(Da);(?)
3) 连(Lian)续(Xu)传(Chuan)代(Dai)观(Guan)察(Cha):(?)永(Yong)生(Sheng)化(Hua)细(Xi)胞(Bao)应(Ying)能(Neng)稳(Wen)定(Ding)增(Zeng)殖(Zhi)超(Chao)过(Guo)50代(Dai),(?)而(?)原(Yuan)代(Dai)细(Xi)胞(Bao)通(Tong)常(Chang)在(Zai)10代(Dai)左(Zuo)右(You)出(Chu)现(Xian)衰(Shuai)老(Lao);(?)
4) 生(Sheng)长(Chang)曲(Qu)线(Xian)测(Ce)定(Ding):(?)绘(Hui)制(Zhi)细(Xi)胞(Bao)倍(Bei)增(Zeng)时(Shi)间(Jian)曲(Qu)线(Xian),(?)永(Yong)生(Sheng)化(Hua)细(Xi)胞(Bao)应(Ying)保(Bao)持(Chi)稳(Wen)定(Ding)增(Zeng)殖(Zhi)速(Su)率(Lu)。(?)
** 功(Gong)能(Neng)特(Te)性(Xing)鉴(Jian)定(Ding)**
为(Wei)确(Que)保(Bao)永(Yong)生(Sheng)化(Hua)细(Xi)胞(Bao)保(Bao)持(Chi)Mü(?)ller细(Xi)胞(Bao)特(Te)性(Xing),(?)需(Xu)进(Jin)行(Xing):(?)
• 免(Mian)疫(Yi)荧(Ying)光(Guang)染(Ran)色(Se)检(Jian)测(Ce)特(Te)异(Yi)性(Xing)标(Biao)志(Zhi)物(Wu)((?)GS、(?)CRALBP、(?)Vimentin)(?)
• 合(He)成(Cheng)酶(Mei)活(Huo)性(Xing)测(Ce)定(Ding)
• 钾(Jia)离(Li)子(Zi)缓(Huan)冲(Chong)功(Gong)能(Neng)检(Jian)测(Ce)
• 对(Dui)视(Shi)网(Wang)膜(Mo)神(Shen)经(Jing)元(Yuan)条(Tiao)件(Jian)培(Pei)养(Yang)基(Ji)的(De)反(Fan)应(Ying)性(Xing)测(Ce)试(Shi)
**细(Xi)胞(Bao)冻(Dong)存(Cun)与(Yu)复(Fu)苏(Su)**
鉴(Jian)定(Ding)合(He)格(Ge)的(De)细(Xi)胞(Bao)系(Xi)按(An)1×(?)10^6细(Xi)胞(Bao)/mL密(Mi)度(Du)冻(Dong)存(Cun)于(Yu)含(Han)10%DMSO的(De)培(Pei)养(Yang)基(Ji)中(Zhong),(?)程(Cheng)序(Xu)降(Jiang)温(Wen)后(Hou)转(Zhuan)入(Ru)液(Ye)氮(Dan)保(Bao)存(Cun)。(?)复(Fu)苏(Su)时(Shi)采(Cai)用(Yong)37℃(?)快(Kuai)速(Su)水(Shui)浴(Yu)法(Fa),(?)离(Li)心(Xin)去(Qu)除(Chu)冻(Dong)存(Cun)液(Ye)后(Hou)重(Zhong)悬(Xuan)于(Yu)新(Xin)鲜(Xian)培(Pei)养(Yang)基(Ji)。(?)建(Jian)议(Yi)前(Qian)3代(Dai)细(Xi)胞(Bao)每(Mei)代(Dai)冻(Dong)存(Cun)5-10支(Zhi)备(Bei)份(Fen)。(?)
**注(Zhu)意(Yi)事(Shi)项(Xiang)**
1. 所(Suo)有(You)操(Cao)作(Zuo)需(Xu)在(Zai)二(?)级(Ji)生(Sheng)物(Wu)安(An)全(Quan)柜(Gui)中(Zhong)进(Jin)行(Xing)
2. 不(Bu)同(Tong)批(Pi)次(Ci)血(Xue)清(Qing)需(Xu)预(Yu)先(Xian)进(Jin)行(Xing)细(Xi)胞(Bao)生(Sheng)长(Chang)测(Ce)试(Shi)
3. 定(Ding)期(Qi)检(Jian)测(Ce)支(Zhi)原(Yuan)体(Ti)污(Wu)染(Ran)
4. 建(Jian)议(Yi)使(Shi)用(Yong)低(Di)代(Dai)次(Ci)细(Xi)胞(Bao)((?)10-30代(Dai))(?)进(Jin)行(Xing)实(Shi)验(Yan)
5. 原(Yuan)始(Shi)细(Xi)胞(Bao)株(Zhu)应(Ying)分(Fen)开(Kai)保(Bao)存(Cun)避(Bi)免(Mian)交(Jiao)叉(Cha)污(Wu)染(Ran)