L*******e
发帖数: 2153 | 1 I am testing transduction of lentivirus (vsv-g) in primary hippocampal
neurons.
I have tried 5ug/ml polybrene for over night, however I have observed
tremendous cell death. : ( Any suggestion for a better way to infect primary
neurons?
Thanks ahead! |
j****x
发帖数: 1704 | 2 有对照吗？
尝试降低vsvg包膜蛋白质粒在转染时的配比，vsvg的毒性对很多细胞比较强。另外就是
尽量提高包装效率，用尽量少的病毒上清感染
primary
【在 L*******e 的大作中提到】
: I am testing transduction of lentivirus (vsv-g) in primary hippocampal
: neurons.
: I have tried 5ug/ml polybrene for over night, however I have observed
: tremendous cell death. : ( Any suggestion for a better way to infect primary
: neurons?
: Thanks ahead!
|
L*******e
发帖数: 2153 | 3 The virus I used is high efficient, it has been tested previously by other
labs on multiple cell lines and stem cells, but not primary rat neuronal
culture. I had a control with polybrene alone, it was toxic. I also
increased the quantity of virus I used, which helped a little bit. The only
thing I haven't tried is to adjust the ratio between virus and polybrene. So
frustrated.....
PS: love your profile photo : )
【在 j****x 的大作中提到】
: 有对照吗？
: 尝试降低vsvg包膜蛋白质粒在转染时的配比，vsvg的毒性对很多细胞比较强。另外就是
: 尽量提高包装效率，用尽量少的病毒上清感染
:
: primary
|
A******y
发帖数: 2041 | 4 Polybrene warns not to used on primary neurons. Do spin transduction maybe? |
L*******e
发帖数: 2153 | 5 what is spin transduction? SORRY, I AM SO DUMB.
maybe?
【在 A******y 的大作中提到】
: Polybrene warns not to used on primary neurons. Do spin transduction maybe?
|
j****x
发帖数: 1704 | 6 如果是这种情况，那自然没啥好说的，降低polybrene的比例甚至完全不加，很多情况
下一样可以感染只是效率降低，我对primary neuro cell不熟，但是在primary
hepatocyte上，spin infection效果还不错
only
So
【在 L*******e 的大作中提到】
: The virus I used is high efficient, it has been tested previously by other
: labs on multiple cell lines and stem cells, but not primary rat neuronal
: culture. I had a control with polybrene alone, it was toxic. I also
: increased the quantity of virus I used, which helped a little bit. The only
: thing I haven't tried is to adjust the ratio between virus and polybrene. So
: frustrated.....
: PS: love your profile photo : )
|
a****d
发帖数: 1919 | 7 You don't have to use polybrene for lentivirus transduction in primary
neuronal culture. If you concentrate the lentivirus, make sure to spin down
the undissolved pellet after re-suspending the viral particle. The
undissolved pellet could be toxic for cells too. |
L*******e
发帖数: 2153 | 8 Do you mean that I should add the virus directly?
down
【在 a****d 的大作中提到】
: You don't have to use polybrene for lentivirus transduction in primary
: neuronal culture. If you concentrate the lentivirus, make sure to spin down
: the undissolved pellet after re-suspending the viral particle. The
: undissolved pellet could be toxic for cells too.
|
L*******e
发帖数: 2153 | 9 what is spin infection? Do you mind sending me your protocol? Thanks!
【在 j****x 的大作中提到】
: 如果是这种情况，那自然没啥好说的，降低polybrene的比例甚至完全不加，很多情况
: 下一样可以感染只是效率降低，我对primary neuro cell不熟，但是在primary
: hepatocyte上，spin infection效果还不错
:
: only
: So
|
j****x
发帖数: 1704 | 10 很简单，就是染毒后细胞培养板在2000rpm（800g？）32度/37度下离心1-2小时，具体
的条件参数根据不用的细胞可能需要调整
【在 L*******e 的大作中提到】
: what is spin infection? Do you mind sending me your protocol? Thanks!
|
L*******e
发帖数: 2153 | 11 with polybrene? or without? I checked some websites, most people still use
polybrene. Do you use it in your case? I guess 1 or 2 hrs incubation of
polybrene is not as toxic as overnight?
【在 j****x 的大作中提到】
: 很简单，就是染毒后细胞培养板在2000rpm（800g？）32度/37度下离心1-2小时，具体
: 的条件参数根据不用的细胞可能需要调整
|
j****x
发帖数: 1704 | 12 without，因为之后并不换液啊。对你的情况先尝试不加polybrene吧，效率可能会降低
，但因为毒性问题也不得不这么尝试一下，没准运气好。假如效率显著降低没法用，就
再试试spin infection with polybrene，然后孵育4-6小时再换液，这样比过夜强点
【在 L*******e 的大作中提到】
: with polybrene? or without? I checked some websites, most people still use
: polybrene. Do you use it in your case? I guess 1 or 2 hrs incubation of
: polybrene is not as toxic as overnight?
|
L*******e
发帖数: 2153 | 13 Thanks SO MUCH. You have been very helpful. Good luck with your experiments/
career. I will update you later. : )
【在 j****x 的大作中提到】
: without，因为之后并不换液啊。对你的情况先尝试不加polybrene吧，效率可能会降低
: ，但因为毒性问题也不得不这么尝试一下，没准运气好。假如效率显著降低没法用，就
: 再试试spin infection with polybrene，然后孵育4-6小时再换液，这样比过夜强点
|
a****d
发帖数: 1919 | 14 That's what I normally do with primary neuronal cells (10 MOI).
【在 L*******e 的大作中提到】
: Do you mean that I should add the virus directly?
:
: down
|
