悬浮细胞专用腺病毒

 

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