包装病毒并不难，一般用293T或293FT做转染，生产病毒，再感染目的细胞，我给你上传一份慢病毒和逆转录病毒的protocol，你看看，很简单。测滴度也不难，病毒感染293T细胞后的48h测，步骤如下： ①. 15万293T细胞48h即可刚好长至90-100％满，此时即可固定细胞，计数确定病毒滴度。 ! X8 E7 R1 e u5 b$ G3 i/ t ②. 将培养基用真空泵吸去部分，务必不要将其吸净，由于293T细胞易飘，固定及DAPI染色整个过程请务必保持293T细胞处于液面之下。用PBS涮3次。加入4％ PFA室温固定30min, 24孔板每孔200ul。% o! x$ O4 p7 g$ Q' O. h/ z
③．用PBT洗2次，每次3分钟。6 c6 z& J. a9 v* B
④．PBS将DAPI 1000倍稀释，每孔加入200ul，避光室温静置5min。2 c7 K" m/ P% N* N% |- ~
⑤．PBS洗2次，每次5分钟，即可在荧光显微镜下计数，取2-3处取平均值。) x7 v2 u, v1 ~
⑥．病毒滴度（IU/mL）=荧光细胞占总细胞数的百分比÷加入的病毒上清体积×1.5×10*5×10*3作者: momocy 时间: 2011-11-17 09:10
Retrovirus / lentivirus infection protocol ( y6 n9 Y. [6 d7 `4 J7 a# VD1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection) # w* L8 ^: r' J/ \
*** Handle HEK293T cells gently to avoid detaching from dishes 2 S: |. o$ f, V: O$ d- C" vD2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction& w. g3 m! t7 |9 \ m2 a/ R
For retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG " \6 p5 @- _4 w, a
For Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG / |1 i3 Y% k8 ` `; H
*** ~6 hours after transfection, change medium to fresh DMEM with 1% FBS supplied with P/S, and incubate at 37°C for ~48hs (two overnights)% @: c g' Y4 h2 X/ E, G
D3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)* N' [5 \0 c9 C8 C! o8 v
D4: Collect viral supernatant from HEK293T cells into 15ml tubes $ q: v w: U7 B! I0 L- ^↓ spin down at 2000RPM for 3mins to remove cell debris ' P* D& Z" F/ T2 o% O2 g. k↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus) 6 J7 f c( f& N& U↓ Wash cell in 6-well plate with 1XPBS4 E8 {. W$ f/ r% i- w, I5 M
↓ Add 1.5ml of filtered viral supernatant to target cells in 6-well plate, then add 8ug/ml of polybrene (by adding 1.5ul of 8mg/ml stock) ; J* B6 |9 |+ @4 M) D/ A↓ Incubate the cells in 6-well plate at 37°C for ~6hrs 7 ]: ^, \; i2 t+ l* n, B* a) v0 Q↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%6 q. s% m0 G1 I. K3 m+ g* o6 |+ L
↓ Incubate at 37°C for overnight $ R2 ~ E4 I; s3 v) R$ f/ ~/ v; S*** After collecting D1 virus, you can add more DMEM with 1%FBS into HEK293T cells and collect D2 virus after incubating overnight. But the D2 virus usually has lower viral concentration.% {- j6 i! X" g# |7 i
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases. / w4 e8 B" T8 I, a1 G*** The leftover of the viral supernatant can be stored at -80°C. ' B) u9 o9 g5 ^8 z; o" LD5: You may start selection with antibiotics if your cells don’t need further infection 5 _* [+ Z$ T1 d6 M. I*** You need to titrate the antibiotic concentration for selection in your cells ahead; u7 f& B& ]2 _8 ]# y 作者: zxcv12345 时间: 2011-11-17 09:57
回复 zhangtang 的帖子# ~0 i" V s* I0 v% n# J& X
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你转染的时候，多转染一个GFP，也就是ＧＦＰ和质粒同时做，算出ＧＦＰ的滴度，然后你质粒的病毒和ＧＦP加的量一样多,这样不就行了么作者: 99牵牵 时间: 2011-12-13 08:47
我的就是这个样子的，本身质粒没荧光，加一个GFP作对照！可是问题是我的包装细胞状态总是不好，包出来的病毒去感染293t荧光很不亮 而且很少 可以告诉我怎么个情况吗？谢谢啦!" I, [& t) r. w& J$ \ 作者: 细胞海洋 时间: 2011-12-13 09:18