Dear Andres,

The killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging. Third, you say that your virus is not able to infect the cells or the resistance is hardly expressed. I think, the reason may be that the titer of your virus is not enough to infect your cells or the MOI is not suitable.

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

Hope this may help you.

You can increase it to 2ug/mL. You should be able to see significant cell death n 2-3  days.

See also links.

http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/in_vivo_preclinical_imaging_ask/biolumcells_ask.xhtml

http://clincancerres.aacrjournals.org/content/suppl/2012/08/29/1078-0432.CCR-12-2130.DC1/meth.pdf

thanks a lot! I´m gonna trying just today.

Hi,

The killing dose for puromycin varies between cell lines, and also among glioblastoma cells. I would suggest that you use a couple of wells to titer the concentration for each cell line to find the optimal dose. We have found that the the killing dose between different glioma cell lines varies between 0.5-4 ug/mL. As mentioned above the cells should start dying after 2-3 days. It might also help to split the cells after a couple of days, since it then is easier to distinguish alive and dying cells.

Good luck!

When you don't know the killiing dose, it is better to start off using lower dozers like 0.5 ug/mL. After 2-3 days, you can check your cells under microscope. If this dose won't kill your non-transfected parental cells (no apoptotic morphology), then increase the dose. The problem with a high dose of puromycin at the beginning of selection is that you might kill the  positively transfected cells

Thanks a lot again for your helpful tips. I´ve just plated cells and tomorrow will start with puro!

If you wish to select for stably transduced cells, you must first determine the minimum concentration of puromycin required to kill your untransduced cell line, perform a kill curve experiment. Typically, adherent mammalian cells are sensitive to concentrations of 2 to 5 µg/ml, while cells in suspension are sensitive to concentrations as low as 0.5 to 2µg/ml.  

I recommend that you test a range of concentrations to ensure that you determine the minimum concentration necessary for your untransduced cell line.

1. Plate cells at approximately 25%-30´% confluence. Prepare a set of 6–7 plates. Allow cells to adhere overnight.

2. The next day, substitute culture medium with medium containing varying concentrations of puromycin as appropriate.

3. Replenish the selective media every 3–4 days, and observe the percentage of surviving cells.

4. Determine the appropriate concentration of puromycine that kills the cells within 10–14 days after addition of antibiotic.

Hi everyone. I am finding a lot of problems. U373 are easy to transform and select with puromycin. In fact, I already have a pure recombinant culture. The problems arrives with the GL261. It is nearly imposible to find a puromycin dose that do not causes an extinction of the culture. It seems that the possibilities are two: the virus is not able to infect the cells or the resistance is hardly expressed. Anyone has experienced similiar problems? I am going to test puromycin concentrations as low as 0.01 ug/ml!!!

Hi Andres,

have you solved the GL261 problem? I'm going to use puro on them and I would like to know if you found the optimal concentration.

Thanks!

Dear Andres,

The killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging. Third, you say that your virus is not able to infect the cells or the resistance is hardly expressed. I think, the reason may be that the titer of your virus is not enough to infect your cells or the MOI is not suitable.

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

Hope this may help you.

Here are some protocols of virus packaging, hope they will be helpful for you.