Dear Jose,

Polybrene indeed increases the efficiency of viral infection, but it is also toxic to some cell lines, and the sensitivity varies from different cell lines. For polybrene accessible cells We recommend the working concentration of polybrene as 6-8μg/ml.

The virus transfection efficiency is influenced by not only polybrene, but also virus titer. You need first make sure that there are many virus particles in your virus concentrations. If your virus titer is below detection limit, it is still difficult for you to determine.

Genemedi got a rich experience in virus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging, if you’re interested.

Hope this may help you.

First possibility is that your virus yield is below the detection limit,

second you probably miscalculated polybren concentration so that it is really toxic to cells,

third check out your filter setting on microscope make sure you use the right filter.

Solution:

repeat the assay and use positive control, then you know what it is that causing the trouble...

What is the envelope on the virus you use? HIV envelope does not do well for an infection after concentration

What promoter drives gfp express? Lenti viral ltr driven gfp has extremely low expression.

if you are worried about Polybrene toxicity, you could add the same volume of it to your cells to check for toxicity. It is quite possible that the virus stock you use had something adverse in it.

Make sure you resuspend your virus stock in complete media and not PBS or dmem alone.

Thanks Salih y Sanath for your replys. About the envelope, it's a HIV-1 pseudotyped vector with a fusion envelope glycoprotein in which the cytoplasmic domain of RV-G was substituted by the corresponding part of vesicular stomatitis virus glycoprotein. The promoter for gfp is MSCV and I resuspended my virus stock in PBS according my protocol..I guess I know what is the problem. I' m performing a new infection and see what happen.

To transduce HEK, you don't need polybrene. But, to enhance the transduction, you can use protamine sulfate instead of polybrene (it's less toxic).

For this cell line, you can directly drop unconcentrated viral surpenatant (filtered 0.45 µM) on the cell.

How have you purified and concentated your rLV ?

If you need some help, we can produce your viral vectors for you and also your cell of interest (http://www.giga.ulg.ac.be/jcms/prod_695820/en/giga-viral-vectors).

I do similar experiments and i use less polybrene because it is really toxic. My lentivirus infects cells without polybrene, but better with polybrene- i think it should be the same with your virus. Resuspension in PBS should be ok- it's according to my protoicoll, too. You should see flourescence the day after transfection if you use hek cells.- Which system do you use? Tronolab 2 generation?

How old is your LV? Maybe it lost effiziecy?Did you freeze it down immediately after concentrating it?

Are your plasmids still ok? Did you check your virus-titer, for example with go-stix?

Hi Jana,

In my case, I've tried with polybrene once and my cells died, but without polybrene transductions were over 80%. I see fluorescence 24 hours post-transfection too. About the system, I follow the system used in this article: http://www.jneurosci.org/content/31/47/17169

The plasmids are ok. I checked the virus-titer by serial dilutions on 293T cells (raw packing supernatant). This week I will perform another production/purification with some recommendations that I received and I could share with you.

Dear Jose,

Polybrene indeed increases the efficiency of viral infection, but it is also toxic to some cell lines, and the sensitivity varies from different cell lines. For polybrene accessible cells We recommend the working concentration of polybrene as 6-8μg/ml.

The virus transfection efficiency is influenced by not only polybrene, but also virus titer. You need first make sure that there are many virus particles in your virus concentrations. If your virus titer is below detection limit, it is still difficult for you to determine.

Genemedi got a rich experience in virus production and titration, you could find more information on https://www.genemedi.net/i/lentivirus-packaging, if you’re interested.

Hope this may help you.