I want to transfect the hippocampal neuron by lentivirus (with GFP), so I produced the lentivirus myself and the virus was resuspend in DPBS(maybe mix some FBS from the media) after concentration.
After dissected the neuron from P1 mouse, I plat about 300,000 neuron in 24 well (the slide was covered by polylysine) with 500uL media (neuron basal+ L-Glu+ B27+ Anti-anti), at the same, I add 2.5uL lentivirus into the media.
I half change the media every two days. In 2 DIV, the neuron was health. However, in 3 DIV, all the neuron was died, even though I can cell mass with GFP.
The neuron without transfect virus were health.
Does anyone ever met this problem?
What is the cause of this problem?
How to solve this problem?
Any suggestion will be appreciated!
Thanks!
I haven't ever seen lentivirus kill cells following transduction...
Some questions: what was the titre of your virus? Did you use sodium butyrate during your virus production? Is the effect dose-dependent (ie if you add more virus, do the cells die sooner, or if you dilute the virus 1:10 do the cells survive)? If you add the PBS only to your cells are they OK? What kind of GFP is your virus expressing?
Hello, Jill
Thanks for your help!
For your questions:
1, The titre of the virus is about 1,500,000 TU/uL.
2, I don't use sodium butyrate during my virus production. By the way,what is the function of sodium butyrate? Did it kill neuron?
3, I don't know whether this virus is dose-dependent, maybe I need check it next time.
4, I don't think the DPBS will kill the neuron, but I will add the DPBS to the cell as a control.
5, The GFP I used was enhanced GFP. However, I don't think this is a problem, my friend in our lab all use this vector to express the same GFP and they don't come across this problem.
1.5e9/mL is quite a high titre - how did you titre the virus (QPCR, fluorescence, FACS)? Did you use ultracentrifugation to concentrate?Try adding DPBS alone to your cells in case there's something wrong with that, and next time add serial dilutions of virus to your cells in case the effect is dose-dependent.
eGFP shouldn't be a problem I don't think, sometimes hrGFP is a bit toxic which I why I asked.
Some people use sodium butyrate during LV production so it's possible that small amounts of it would be left in the final prep, but you don't so it's not that.
You could have contamination from the HEK cell debris and FBS in your virus solution. If you purified just by ultracentrifugation (without sucrose cushion), you will always have it. In the most of cases it works fine, but primary neuronal cultures are very sensitive and easy-to-die. You could first try just to centrifuge your resuspended lenti in a bench-top centrifuge at max speed for 1 min. The majority of debris will be precipitated. Virus will stay in the solution. If it does not help, then you will have to do a second ultracentrifugation step with sucrose cushion.
Hey,
getting a tire of 1500000Tu/µl is really high! BUt further more this does not mean that you have only this particles, because you can onnly measure the "active" viral particles This means you have a lot of inactive viral particles and you put a lot of wate on your cells. Furhter more your MOI seems quite high. You could half it or exchange the SN 6h past transduction.
Sodium butyrate is epigenetically triggering virl promoters like CMV and by this is thought to increase virus production
Regards
Sven
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