Hi All,
I have done a first run of infection with several lentivirus containing different shRNA this week. I am about to add puromycin but just realized that I forgot to add polybrene!
I will start a new run of lentivirus infection with polybrene next week, but does anyone know if lentivirus (envelop is VSVG) can infect HCT116, T24, and A549 cells without polybrene?
Thanks,
Lyra
Hi Lyra,
It is definitely possible to transduce your cells without the presence of polybrene or any other polycation. See this publication (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456965/pdf/nihms380409.pdf). However your efficiency of transduction is massively higher with the adition of polycation. It can also make a difference which serum you grow your cells in.
Nonetheless, if you are going to select with puromycin you will find positive cells in your dish. I would go ahead with the selection
Cheers,
Jeroen
Thank you so much for your reply, Jeroen! I will start the selection this weekend and let you know how that goes! ;-)
It will transduce the cells but transduction efficiency may be low without polybrene. I think its worth to go ahead with puromycin selection.
I agree with both the answers above. However, here's a small point i'd like to add. All the cells you mentioned are cancer/carcinoma cell line. I've spent quite sometime trying to get a possible clone with LV in primary/semi primary cells like HUVECs or HFFs. Both of these do not react well to polybrene while without polybrene the efficiency of transduction is ridiculously low. What I did instead was to up the viral titre a bit and incubate for a day or two at most, following which the virus infected media was removed and the cells were cultured in their respective medium. The loss of cells was high for sure but i also got one ore two clones that i could propagate, at least for a while.
Not sure if this helps but i always wonder about the primary nature of these cells because some of these clones can actually be propagated for quite a while before the stop dividing.
Best wishes,
Roy
Thank you so much Shivam & Roy!
Up the virus titer is a good point. Today I will go & check out those cell. Will let you guys know if I get any surviving cells in Puromycin ^_^
Have a great day!!!
Lyra
Hi All,
I have checked my puromycin-selected plates yesterday. I found that I can get lots of stable cells from the ones that are infected with higher titer virus. For the ones that with lower virus titer, I got some stable cells but most are dead (so not infected by virus).
I will start another run of puromycin selection tomorrow (same virus, same amount of virus, same cells, but with polybrene this time) and will let you guys know how it compare with this earlier batch (without polybrene).
As a side note, it seems that the plasmid concentration affect virus titer quite a bit. Although I add same amount (3ug) of plasmid for generating the virus (lipofectamine3000), the ones that have low plasmid conc. (~100ng/ul) gives lower virus titer than the one with higher plasmid conc. (~300ng/ul).
Thank you guys again for your help!
Lyra
Hi All,
I checked the puromycin selection result of my 2nd infection (with polybrene).
I really cannot see much difference in the amount of cells that survive the selection comparing to without polybrene (well, maybe a little bit more surviving cells for the viruses with lower titer in HCT116 and T24 cells).
Best,
Lyra
I think all depends on the virus titer.Did you concentrated the virus or just the soup.??
I am getting a good infection without adding polybrene.But I concentrated the virus using ultracentrifugation.
I am also working with HCT116. Did you count the surviving cells and find it comparable between with and without polybrene?
Thanks.
Hi Zhu,
I didn’t count the surviving cells, but they look similar when inspected by eyes.
Best,
Lyra
Hi, I am working on HCT116 and HT29 cells. May I know what was the concentration of puromycin you used for the selection? Need it as a reference. Thank you.
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