I had injected retrovirus containing pBabe.puro.cmv and pcl ampho to the F1 B16 and then after 24 hours and 48 hours I did puromycin selection, The First days and second days the cell decrease gradually and the thirds days all the cells died, is there posibilty the virus accepted to the cells then several days rejected and out to the cells? please give suggestion about this.. thansk very muchI had injected retrovirus containing pBabe.puro.cmv and pcl ampho to the F1 B16 and then after 24 hours and 48 hours I did puromycin selection, The First days and second days the cell decrease gradually and the thirds days all the cells died, is there posibilty the virus accepted to the cells then several days rejected and out to the cells? please give suggestion about this.. thansk very much?
The cells you are working with may be very sensitive to puromycin. The concentration of puromycin added to the cells may be too high. For each new cell type used, it is recommended that a puromycin titration be performed to determine the lowest concentration of puromycin needed to efficiently select transfected or transduced cells. Also, be sure to wait 48 hours post-transduction before applying antibiotics to the cells.
How should a Puromycin titration (kill curve) be set up? Look here http://info.agscientific.com/blog/bid/160008/The-stuff-you-should-know-about-puromycin
To make sure that your virus preparation process is ok, you may include the pBabe EGFP control, the infected cells will give you green fluorescence light. (If your institute is equipped with fluorescence microscope.)
thanks very much, about concentrace puromycin we already had done puromycin titration for B16 cells, and the preparation retrovirus had already include EGFP. how about the titer retrovirus? is there corelation between titer retrovirus with infected efficiency to the cell?
I used Empty vector without gene or protein of interest, how about the promoter? is it posible? may be the promoter which is not suitable with B16 cells
the first I seeded 6.105HEK 293T 24 hours later transfected plasmid p.Babe.puro cmv and plasmid pcl ampho with PEI and PBS (DNA:PEI = 1:3) then 12 hours change the medium and 48 hours after change medium harvest the virus used filter 0,45 µl
Hi Dewi,
I have been doing transfection/infection for a while now and this problem happened to me sometimes. Several possibilities can be raised:
- your new plasmid prep may not be as good as you think. You can put it on gel and see the purity of your plasmid. Be sure that you have a nice supercoiled purified plasmid, it is the most efficient.
- it happened that my HEK started to float. They might have been contaminated by mycoplasma or I kept them in culture too long. In any case, your HEK might be in a condition where transfection or virus synthesis is not correct. You can make a test to verifiy the efficiency of your transfection, for example by checking if the HEK express the GFP you transfect. In any case, if I were you, I would thaw a new batch of HEK. This worked several times for me (and I couldn't determine what happened).
- finally, be sure about the reagents you use for transfection. I guess you transfect with HBS and CaCl2. Check the pH of HBS or prepare new stocks (even for the water).
I hope this will help. This can of problem can follow you for weeks so if you don't find any solution, don't hesitate to get rid of everything (including your cell lines) and thaw/prepare new reagents.
Good luck!!
Mathieu
I have never titered my retrovirus. I used to take the supernatant and filter it directly on the cells I want to infect. If your HEK are well transfected, which is the first step to verify, you should have a high concentration of virus. You can decrease a bit the volume of media you put on your HEK to concentrate your viruses. But it should work fine
Did you light your target cells up with green fluorescence when you infected the cells with the EGFP expressing virus?
when I prepared retrovirus I used 2 plate, one plate contained p.babe puro cmv and pcl20c.cmv.flag EGFP as my control transfection and the other plate contained p.babe.puro cmv and pcl ampho. Should I put EGFP to all dish? Thanks very much
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