I have tried PEG-it, wasn't a big fan. What's the volume of of supernatant that you are generating? Do you have access to a decent tabletop centrifuge?

hi

You have come up with a clever question for surveying common kits.

good luck

Jill Muhling Hi Jill, thank you for your answer. I am generating up to 50 ml of supernatant and my tabletop centrifuge goes up to 3000g. Could you concentrate the lentiviruses with the PEG-it kit and perform the subsequent transduction with no problems?

Reza Hadavi Hi. My question is pretty clear. I am asking people that have used any of these kits to tell me their opinion on it since I want to order the one that is the most reliable.

Hi Alejandro, I found that the PEG-it kit worked OK but I often ended up with particles in the final prep which adversely affected the cells. It would certainly be worth a try, see if the company will give you a free sample.

You can also try pelleting the virus for 24 hours at 3000g - overnight at 3500g works with some lentivirus pseudotypes so it would be worth trying before you go spending money on kits.

I've used PEG-it and re-suspended the final particles in PBS, then kept frozen in aliquots at -80 Celsius. I then used these cleaned up particles on primary hippocampal neurons from mice and others in my lab have as well to do imaging experiments among other things. It worked well for all of us, we were using it to overexpress or knock out microRNA. Hope that helps.

Jill Muhling Thank you for sharing your experience with the kit! Did you see an important drop in viability after transduction? I need that they keep an acceptable viability before sorting them by FACS.

Amrit S Boese This is exactly what I wanted to know, since I will also use them in murine cells for imaging purposes. Did you experience any problem with the cells after the transduction? Thank you so much, it was very helpful :)

With PEG-it? I did see reduced viability, but I was working with a lentivirus that produced very poorly so I was obliged to add quite a bit of virus to the cultures. If you have a good concentrated titer you would possibly not have that issue.

Alejandro Sanz Pérez :Our lab published this paper where it was used, there seemed to be minimal impact to the cells as per a CytoTox 96 assay as described: Article MicroRNA-16 targets mRNA involved in neurite extension and b...

Jill Muhling : we had really good production of virus so likely that contributed to our success, I can see how high levels could hurt cells. For my personal work, I switched out to using a different delivery system as I was concerned about the antisense strand of the miR being intact with a plasmid expression system.

We use PEG8000 (storage concentration 40% in PBS) to concentrate the lentiviral particles. The work concentration of PEG is 10%. The PEG8000 (40%) was added into harvested supernatants after HEK-293T co-transfection at 1/3 volume. Rotation at 4 °C overnight. On the next day, the virus particles were harvested by centrifugation at 4 °C 4000 rpm for 30 min and then transfected the cells.