It could be the polybrene. I have transduced MSCs at high efficiency without it. Try spinning the plated cells w/ lentivirus. 32 deg C, 1200 x g, 30 min

Agree with Artem. Try to titrate the polybrene.

Hi Sara, probably it could be the polybrene. I have trasduced murine bone marrow stem cells using polybrene to 5 ug/ml. My protocol suggests to add to virual supernatant  polybrene and Hepes to 10mM, but I performed transfection successfully only with polybrene 5ug/ml. As Artem suggests, try spinning cells at 2100 rpm for 45 minutes at room following  75 minutes incubation at 37°C. after this incubation  remove viral supernatant. 

It could be because of either transduction or polybrene (or even both). I would titrate both to find the optimal concerntration for the cells to survive. As people above suggested spinning the cells with viral supernatant might also help in survival. Based on the intensity you can even facs sort for low,medium and high RFP intensity and see if at least low RFP cells survive.

Thank you very much for all your answers. I think I will start to titrate the Polybrene and even try it without it. At the beginning of my experiments i titrated the virus concentration  too.

to Christopher: Especially to you a big thank you!!!

• At first i want to say, that I am sure to have proliferating hMSC's because I passaged them before and did other working experiments with them (si RNA transfection etc). so this should not be the problem. I got the cells from a cooperation partner and the youngest passage I have is P5. The lentiviral experiments i did with P7...so maybe they were too old. But it does not look like that.
• "Cell concentration is important, you your transduced cells plated at a density that gives you best the yield without crowding which leads to cell cycle arrest, 80% is good."     This could be a point. maybe the were to dense. Thank You.
• "One question, you say are using lentivirus, but the promoter is CMV not the LTR element. I have not done these studies with my own hands so I might be missing something, but do RV expression vectors have CMV promoters?? I have seen dual promoters in vectors that express two portion in tandem, so maybe this is a CMV promoter for the RFP as second protein??"  Maybe I explained it in the wrong way. The CMV promoter is for the crimson. the target gene has an U6 promoter. I attached you a plasmid map to have a look at it, if you like :) of course there are the LTR elements.

When I like to do the spin transfection without Polybrene I will need a centrifuge where I can directly put my 6-well plates inside, didn't I? Is there any other possibility? Because our S2 part of the laboratory is very small and we're just having centrifuges for eppendorf tubes (1,5 ml) and falcons/greiner tubes (15 or 50 ml).

Thank you very very much!

thanks Xue-jun.

Hey guys,

at the end I got to the bottom of the problem. In my case the PEG6000 precipitation was the critical step. In recent experiments I just leave the precipitation out of my protocoll and just infected the cells with HEK-medium including the virusparticel, and now everything is fine. even with polybrene. 

hi sara!

could you please tell me  the steps and the exact materisl you are using i would really appreciate that, I have problem with MSC transduction too, I did it at a range of polybren and virus concentration , but it did not work!

I'd like to add something to this topic:

I really recommend using the ultracentrifugation method to concentrate viral particles. For me that is the most effective and most gentle way to infect MSC.

Concentration of the virus (eg. ultracentrifugation) eliminates the need to use polybrene. Alternatively, using retronectin (a recombinant fibronectin fragment) may work as well but it costs some €s or $s