汉恒悬浮细胞专用腺病毒特点
1.XUANFUXIBAOZHUANYONG
HANHENGSHENGWUKAIFADEXUANFUXIBAOZHUANYONGXIANBINGDUZHENDUIXUANFUXIBAODEXIBAOMOTEXINGJINXINGLEXIANBINGDUDEGAIZAO,BINGDUGANRANTIXIYEJINXINGLEXIANGYINGDEYOUHUA,JIDAGAISHANDUIXUANFUXIBAODEGANRANNENGLI。DUITIEBIXIBAODEGANRANNENGLIWUYINGXIANG,KEYITONGYONG。
2. CHAOQIANGGANRANXIAOLV
HANHENGXUANFUXIBAOZHUANYONGXIANBINGDUDUIJurkat、K562DENGZHONGDUOXUANFUPEIYANGXIBAOXIGANRANXIAOLVJIEJIN100%,DUIXUEYELAIYUANYUANDAIXIBAO,RUCD4+ TXIBAOGANRANXIAOLVKEDA60%YISHANG。
腺病毒的细胞感染特性
1)XIANBINGDUBUCHARUXIBAOJIYINZU,BUXINGCHENGWENDINGZHUANRAN;
2)XIANBINGDUDESHUNSHIBIAODANENGLIYIBANWEI2-4ZHOU,XIBAOZENGZHIYUEKUAI,BIAODASHIJIANYUEDUAN;
3)YOUYUBUHUISUIJICHARUSUNSHANGJIYINZU,XIANGBIJIAOMANBINGDU,XIANBINGDUDUITIWAIPEIYANGDEMUDEXIBAODUXINGJIAOXIAO,YINCIKEYIYONGJIAOGAOMOILAIGANRANMUDEXIBAO。
4)GAODIDU:HANHENGSHENGWUTIGONGDEXIANBINGDUYABOappGUANWANGDIDUJIAOGAO,YOUYUCAIYONGLEHANHENGTEYOUDETIXI,YONGYUXIBAOGANRANJIBIEDEXIANBINGDUYIBANKEYIDADAO1010 PFU/mlDIDU。
腺病毒细胞实验策略
YOUYUXIANBINGDUDEFEIWENZHUANXING、DILITIXIBAODUXINGYIJIGAODIDU,YONGXIANBINGDUGANRANXIBAODEYIBANCELVESHI:XIANKUOZENGZULIANGDEMUDEXIBAO,RANHOUYONGXIANBINGDUGANRANMUDEXIBAO,DAIMUDEJIYINBIAODAHOUZHIJIEJINXINGHOUXUDEXIBAOGONGNENGXINGSHIYAN。YIBANBUHUIYONGXIANBINGDUXIANGANRANXIBAOHOUZAIDUIXIBAOJINXINGERCIKUOZENGFANGDAHUAPEIYANG。
悬浮细胞专用腺病毒感染实验相关知识点
1)XUANFUXIBAOZHUNBEI:XIANBINGDUJIANWENZHUANXIBAOXIQIAN,QINGQUEBAOMUDEXIBAOZHUANGTAILIANGHAO、WUZHIYUANTIWURAN。XIANBINGDUGANRANDESHIHOUXIBAOHUIHELVWEI40%~60%JIANWEIYI。
2)GANRANXIBAOZUIJIAMOIDECEDING:MOI(GANRANFUSHU)SHIZHIMEIGEXIBAOGANRANDEBINGDUSHU。
ZHU:XIANBINGDUDUIYUBUTONGZHONGLEIBUTONGLAIYUANDEXIBAO,QIZUISHIMOIGEYOUCHABIE。DUIYUXUANFUXIBAO,XIANBINGDUMOIYIBANYI30, 100, 300, 1000, 2000DEBILIDIZENGJINXINGTIDUMOSUO,XIANGBIYUTIEBIXIBAO,XUANFUXIBAODEXIANBINGDUMOIXUQIUGENGGAO,MOITIDUMOSUOJIANYIYONG96KONGBANJINXINGYIJIESHENGBINGDUHEXIBAO。
3)MOIDUIYINGBINGDUTIJIDEHUANSUAN(FEICHANGZHONGYAO!)
感染时细胞数(一般以传代计数细胞数×2计算)×MOI=病毒个数;则加入的病毒体积数=病毒个数/病毒滴度
比如第一天传代1×104细胞到96孔板一个孔,第二天长到2×104细胞,按照MOI=500计算,需加入病毒总量为2×104×500=1×107个病毒粒子,按照腺病毒1×1010 PFU/ml的滴度,则加入的病毒体积数为(1×107)/(1×1010 PFU/ml)=1×10-3 ml=1 μl。
腺病毒感染细胞步骤
1.GANRANQIANZHUNBEI
1)ANSHIYANXUYAOJIANGXUANFUXIBAOPUBAN(BIRU96KONGBAN)37 ℃ PEIYANGGUOYE。XIBAOSHUYIDI2TIANMIDUYUE40%~60%JIANWEIYI。
2)CONGBINGXIANGQUCHUXUANFUXIBAOZHUANYONGXIANBINGDUBINGZAIBINGSHANGHUANXIANRONGHUA,DAIWANQUANRONGHUAHOUKAISHIBINGDUGANRANSHIYAN。
2. MUDEXIBAODEGANRAN
XIQUXIBAOYUANYOUPEIYANGJI,JIARUXINXIANPEIYANGJI,XUANDINGMOIZHIHUANSUANDUIYINGDEBINGDUYUANYETIJI,BINGANTIJIJIANGBINGDUYUANYEJIARUXIBAOZHONG,YAOYUN。FENGHAOKOU,FANGRUPINGJIAOLIXINJIHOU,DISULIXIN1.5h,RANHOUFANGRU37℃PEIYANGXIANGZHONGJIXUJIGANRAN4XIAOSHI。
1) 37 ℃PEIYANGXIANGZHONGGANRANWANBIHOU,QUCHUXIBAO,XIQUHANBINGDUDEPEIYANGYE,HUANSHANGXINXIANPEIYANGJI,JIXU37℃PEIYANG
2)GANRAN48XIAOSHIHOU,YINGGUANGXIANWEIJINGJIANCEGFP/RFPDENGYINGGUANGBIAODAXIAOLV。DUIYUBUFENXUANFUXIBAO,YINGGUANGBIAODAJIAOMAN,XUDAI96XIAOSHIHOUCAIHUIYOUMINGXIANYINGGUANG
ZHU:WEIXINSAOMAGUANZHUHANHENGSHENGWUGONGZHONGHAO,GUANKANHANHENGSHENGWU-XIANBINGDUXIBAOGANRANSHIYANCAOZUOJIAOXUESHIPIN
悬浮细胞专用病毒感染实例
病毒:悬浮细胞专用腺病毒Ads-GFP,1×1010 PFU/ml
感染细胞:Jurkat、K562、HL-60、L2110
感染方式:MOI=500
检测方法:感染72H后荧光显微镜检测
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