原代 T 细胞分离培养及病毒感染操作手册文件下载

 

原代 T 细胞分离培养及病毒感染操作手册

 

HANHENGSHENGWUDUJIAYANFASHIYONGYUYUANDAITXIBAODEBINGDUYOU:MANBINGDU、NIZHUANLUBINGDU(mTRvHEhRv)HEXUANFUXIBAOZHUANYONGDEXIANBINGDU(Ads)。QIZHONGNIZHUANLUBINGDUGANRANXIBAOJUYOUWUZHONGTEYIXING,SHIYONGXIAOSHUYUANDAITXIBAODEWEImTRv,SHIYONGRENYUANDAITXIBAODEWEIhTRv。NIZHUANLUBINGDUKEYIYONGLAIGOUJIANWENZHUANXI。ZHUANYONGXIANBINGDUAdsBUQUFENWUZHONG,DUIREN、XIAOSHUDENGWUZHONGDEYUANDAITXIBAOJUNJUYOUHENGAODEGANRANXIAOLV,LINGWAI,AdsYEFEICHANGSHIYONGGANRANQITAXUANFUXIBAO,RUJurkat,K562,HL-60HEL1210DENG,AdsZHUANYONGXIANBINGDUBUSHIHEYONGLAIGOUJIANWENZHUANXI,XIANGQINGQINGCANKAO“HANHENGXUANFUXIBAOZHUANYONGXIANBINGDUCAOZUOJISHUWENDANG”。

 

 

一. 小鼠脾脏细胞悬液制备

 

1. TUOJINGCHUSIXIAOSHU,FUBUCHAOSHANGZHENGTIWEIBAOLUXIAOSHUYOUCEFUBU。

2. 75%YICHUNXIAODU,JIANKAIFUBUQIEQUPIZANG。

3. JIANGPIZANGFANGRUYUXIANFANGZHIHAOcell strainer(FALCON,352350)BINGYIJIARU5ml WUJUNPBSDELIUKONGBANHUOPEIYANGMINZHONG。

4. YONGWUJUNZHUSHEQIBINGCHONGFENYANMOPIZANG。

5. QIDIAOcell strainer,QINGROUCHUIDAXIBAOZHICHONGFENZHONGXUAN,SHOUJIDAOYIWUJUN15 mlLIXINGUANZHONG,YU4℃ 400g,LIXIN5 min。

ZHU:YIDANQUCHUPIZANG,QINGZHIYUBINGSHANGCAOZUO。

6. JIANGDI5BULIXINHOUQISHANGQINGDEPIZANGXIBAOYONG5 mlWUJUNACKLIEJIEYEQINGROUCHONGFENCHUIDAHUNYUN,YUBINGSHANGLIEJIE5 min。

ZHU:ACKLIEJIEYEPEIFANGRUXIABIAO,PEIHAOZHIHOUYONG0.22 μmDELVMOGUOLV,4℃BAOCUNBUCHAOGUO1YUE

配制 500 ml 1´ACK buffer

NH₄Cl

4.1 g

KHCO3

0.5 g

0.5 M EDTA-2Na

100 ml

7. LIEJIEJIESHUHOUJIARU10 mlWUJUNPBSZHONGZHILIEJIEFANYING,YU4℃ 400gLIXIN5 min。

8. 弃上清,用10 ml无菌PBS洗一次,弃上清后直接加入培养基重悬计数,一只脾脏可得3-10´107个细胞。

ZHU:RUGUOYAOJINXINGXIAYIBUDEFENLICD4+TXIBAOCAOZUO,QINGZHIJIEANZHAOSHUOMINGSHUJIANGXIBAOZHONGXUANZAIHESHITIJIDEPBSZHONG。

 

 

二. 人PBMC细胞制备

 

1. QUYI15 mlLIXINGUAN,JIARU2 mlYIYONGKANGNINGJICHULIGUODERENXUEYEYANGPIN,ZAIJIARUDENGTIJIDEPBSJINXINGXISHI(ZHONGTIJI4 ml)。

ZHU:RUOWEIJINGGUOCHULIDENONGSUOXUEYEYANGPIN,QINGANZHAOXINDEXISHIBEISHUHELIXISHI。WEILEBAOZHENGFENLIXIBAODEHUOLIHEXIAOLV,QINGSHIYONGXINXIANXUEYE,XUEYEYANGPINQINGYU18-20℃BAOCUN。

2. SHANGXIADIANDAOHUOYONGQIANGHUNYUNYANGPIN,BEIYONG。

3. SHANGXIAFANZHUANQUEBAOFicoll-Paque PLUS (GE)FENLIYECHEDIHUNYUN,QUDIAOJUBINGXIPINGGAI,SHIYONGZHUSHEQIZHENTOUCHARUPINGSAIXIQU3 mlFENLIYE。

ZHU:FENLIZHIQIANQINGJIANGFicoll-Paque PLUSFENLIYEYUREZHI18-20℃。

4. QUYIXINDEWUJUN15 mlLIXINGUAN,JIARU3 ml BUZHOU3ZHONGQUCHUDEFENLIYE。

5. XIAOXINJIARUXISHIHAODE4 mlXUEYEYANGPIN,BUYAODAPOFicoll-Paque PLUSYEMIAN。

ZHU:KEJIANGLIXINGUANQINGXIEZHIYUSHUIPINGTAIMIANCHENG30°,HUANMANYANGUANBIJIARUXUEYEYANGPIN。

4.JIANGLIXINGUANXIAOXINSHUZHIFANGRULIXINJINEI,YU18-20℃,400gLIXIN30-40 min。

ZHU:TEBIEZHUYIJIANGLIXINJIDEJIANGSUJIASUDUDIAODAO0。

6. XIAOXINQUCHULIXINGUAN。CISHIYETIFENWEI3CENG,ZHONGCENGWEIFENLIYECENG,ZUIXIACENGWEIHONGXIBAO,ZUISHANGCENGWEIXUEJIANG。WEIYUZHONGCENGKAOJINXUEJIANGCENGDEBAIMOCENGJIWEIDANGEHEXIBAOCENG,XIAOXINXIZOUXUEJIANGCENG,BUYAODADONGBAIMOCENG。

ZHU:KEXICHUZUISHANGCENGDEXUEJIANGCENGBAOCUNYIBEIHOUXUSHIYONG。

7. YONGWUJUNYIYEQIANGJIANGBAIMOXIDAOYIXINDELIXINGUANZHONG。

8. YONGSANBEITIJI(YUE6 ml)DEWUJUNPBSZHONGXUANBUZHOU7ZHONGDEPBMC,ZHONGXUANSHIYONGQIANGQINGROUCHUISAN,YU18-20℃,400gLIXIN10-15 min,ZHONGFUYICI,ZUIHOUYONGPEIYANGJIZHONGXUANBEIYONG,DUOYUDEPBMCKEYIDONGCUNJIXUSHIYONG。

TEZHU:ZAIJIAOGAODELIXINSUDUXIA(400-500g)KEYITIGAOPBMCDEDELV,TONGSHIXUZHUYIZAIJIAODIDELIXINSUDUXIA(60-100g)KEYIFENLIDIAOBUXUYAODEXUEXIAOBAN。LINGWAIRUSHANGCAOZUOHEBILISHIYONGYUGENGDATIJIDEFENLI。

 

 

三. CD4+ T 细胞体外刺激

 

1. ANZHAOXIAOSHUHUORENYUANCD4+ T XIBAOFENLIkit (Thermo) SHUOMINGSHUFENLIDEDAOCD4+ T XIBAO。

2. QUSUOXUTIJIDEPBS,ANZHAO5 μg/mlDEZHONGNONGDUJIARUAnti-mouse CD3eHEAnti-mouse CD28KANGTI,500 μl/KONGBAOBEI Non-Treated 24-well plate,SHIWENFANGZHI4h,XIDIAOKANGTIXUANYE,JIARU500 μl 1% BSA(PBSPEIZHI)SHIWENFENGBI30 min,FENGBIJIESHUJIARU500 μl PBSXIKONGYICI。

ZHU:QINGZAIFENLIXIBAODETONGSHIZHUNBEIHAOPINGBAN,BAOKUOHOUXUGANRANSUOXUDELIANG。

3.将分离得到的CD4+ T按照浓度5´105 cell/ml重悬在完全T细胞培养基中(1640, 10% FBS, 1% penicillin-streptomycin, 50 mM β-mercaptoethanol, 100 U/ml IL-2),将细胞加入步骤2中包被处理好的平板,2 ml每孔(1´10cell/well),然后放入37℃ CO2培养箱刺激培养48h。细胞在刺激24h后体积略微增大,进入活化状态,刺激48h后可进行病毒感染。

SI. CD4+ TXIBAOGANRAN(YIGUOBIAODAGFPDEBINGDUWEILI)

1. SHOUJIHUOHUAHAODECD4+ TXIBAO,400gLIXIN5min。YONGTXIBAOPEIYANGJIZHONGXUANJISHUBEIYONG,ZAISHOUJIDEGUOCHENGZHONGFAXIANYOUXIEXIBAOHUITIEBI,CIWEIZHENGCHANGXIANXIANG,KEFANFUCHUIDAZHITIEBIDEXIBAOYECHENGXUANFUZHUANGTAI。

2. 另取一Anti-mouse CD3e和Anti-mouse CD28抗体包被好的24孔板,每孔加入2-5´105的细胞(若为96孔板,加入2´104细胞/孔)。若考虑长期表达目的基因,按MOI=50-100加入汉恒生物T细胞专用逆转录病毒mTRv-GFP或者hTRv-GFP,注意鼠源和人源原代T细胞所适合的逆转录病毒种类差别;若考虑瞬时(一周以内)表达目的基因,可按MOI=500-1000加入汉恒悬浮细胞专用腺病毒Ads-GFP。同时加入polybrene至终浓度6 ug/ml,最终感染体积为500 ul,加入病毒后轻轻吹打混匀。

ZHU:JUTIMOIXUANZEKEZAI96KONGBANZHONGYUSHIYANJINXINGTIDUMOSUO,DUIYUNIZHUANLUBINGDUJIANYISHIYONG10,30,100DETIDUJINXING,DUIYUXIANBINGDUJIANYISHIYONG500,1000,1500DETIDUJINXINGMOSUO。

3. 24-well plateYU1000 g SHIWENLIXINGANRAN90 min。LIXINJIESHUHOUJIANGPINGBANFANGRU37℃ CO2PEIYANGXIANGGANRAN6 h。

ZHU:LIXINJIESHUGUANCHAXIBAOFENSANZHUANGTAI,CHUFEIXIBAOQUANJUJIZAIYICHU,FOUZEBUJIANYICHUISANXIBAO。

4. GANRANJIESHUHOUQUCHUPEIYANGBAN,XIAOXINXIDIAOGANRANKONGZHONG350ulDEPEIYANGJI(70%),JIARU1.85 mlDETXIBAOWANQUANPEIYANGJIBUZUTIJIWEI2 mlBINGCHUIDAHUNYUN。

5. KEXUANBUZHOU:BINGDUGANRAN24hHOU,XIAOXINXIDIAOPEIYANGJI,ZAITONGYIPEIYANGMINZHONGANZHAO2-4BUJINXINGBINGDUFUGANRANCAOZUO。

6. JIANGXIBAOFANGRU37℃ CO2PEIYANGXIANGJIXUPEIYANG,2-3TIANHOUKEGUANCHAYINGGUANG,ZUOLIUSHIJIANCEGANRANXIAOLV。ZHENGCHANGQINGKUANGXIA,HANHENGSHENGWUTIGONGDETXIBAOZHUANYONGNIZHUANLUBINGDU(mTRvHEhTRv)HEXUANFUXIBAOZHUANYONGXIANBINGDU(Ads)GANRANYUANDAITXIBAODEXIAOLVDAYU50%。

ZHU:GANRANHOUCIJIDEXIBAOZENGZHIJIAOKUAI,QINGZHUYIGUANCHAXIBAOMIDUBINGSHIQIWEICHIZAI1´106/mlZUOYOU。CIWAI,RUGUOXUYAORANGXIBAOZENGZHIJIAOZHANGSHIJIAN,WEILEFANGZHIQIGUODUHUOHUA,KECHEDIAOKANGTIDECIJI。

 

 

五. 原代T细胞病毒感染结果(见下)

 

 

汉恒原代T细胞感染实例——使用悬浮细胞专用腺病毒ads感染

 

悬浮细胞专用腺病毒Ads感染小鼠原代CD4+ T细胞

 

 

XIBAO:XIAOSHUYUANDAICD4+ TXIBAO

CIJIFANGSHI:CD3/CD28KANGTIHEIL-2JIHUO48h

BINGDU:Ads-GFP, 1×10^10 PFU/ml

SHIYONGFANGSHI:MOI=500

 

 

悬浮细胞专用腺病毒Ads感染人原代CD4+ T细胞

 

 

XIBAO:RENYUANDAICD4+ TXIBAO

CIJIFANGSHI:CD3/CD28KANGTIHEIL-2JIHUO48h

BINGDU:Ads-GFP, 1×10^10 PFU/ml

SHIYONGFANGSHI:MOI=100

 

 

汉恒原代T细胞感染实例——使用慢病毒和逆转录病毒感染

 

原代T细胞专用病毒-慢病毒感染人原代CD4+ T细胞

 

 

XIBAO:RENYUANDAICD4+ TXIBAO

CIJIFANGSHI:CD3/CD28KANGTIHEIL-2JIHUO48h

BINGDU:hTLv-GFP,1*10^8 TU/ml

SHIYONGFANGSHI:MOI=50

 

原代T细胞专用病毒-逆转录病毒感染小鼠原代CD4+ T细胞