I`m doing a lentiviral transduction of HaCat cell line and one of my viruses contains puromycin resistance. I`ve chosen the optimal concentrations of the antibiotic on non-resistant cells and now i`m doing stable clones selection.
The question is how stable puromycin is in the solution? i`ve diluted it to 10mg/ml in PBS and store at -20 (supplier recommendations), and for working concentration i`ve diluted it in DMEM to 10mkg/ml and store at +4.
How long may i use the 10mkg/ml solution? Is it light-sensitive?
Hi Alena, you will find the concentrations to use at http://www.lifetechnologies.com/ca/en/home/life-science/cell-culture/transfection/selection/puromycin.html?s_kwcid=AL!3652!3!26107407268!e!!g!!puromycin&ef_id=U-YA-wAABJvHc3ZA:20140821142359:s. As for the stability you can store the solution at -20C for up to 2 years. Needless to say that keeping things in the dark is always the best, a condition that is given in the freezer anyways. For more information you might want to resort to https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/2/p9620dat.pdf
Thank you, Ludger! Yes, i`ve found the datasheet, but there is no information about the light sensitivity. Your answer seems reasonable and i`ll store the dissolved solution protected from light. Maybe you have any idea about how long i may use the 10mkg/ml solution before the degradation of puromycin? like a week, month or half of a year?
You are welcome. AS I mentioned above, Puromycin is sold as a solution. Recommended storage is -20C. It is supposedly good for 2years. So you should be fine storing it at the same condition for about a couple of weeks as well. As for the selection media, you do not want to overdo your use. Once the cells are selected you should be fine. I would occasionally repeat the treatment to be on the safe side. Worked for me.
I found some where (if necessary I can look it up again) that puromycin is very stable if refrigerated. BUT at 37°C it is relativly unstable. Meaning that you should not keep the media to long in the waterbath e.g. and change of medium if not all cells died as they should. I faced once the problem with variation of puromcyin killing efficiency...
Thank you Irene! I haven`t kept the media with puromycin at rt for mor ethan half an hour, but my cells were living in the incubator at 37C in the same medium for 3-4 days. I finally decided to increase the concentration of puromycin due to its possible overtime degradation in the refrigerator (because my HaCat cells were dividing faster than dying so i wasn`t able to perform the selection well). After increasing the concentration to 0.7-0.8 mkg/ml the selection dynamics was ok. And i`ve kept the working puromycin solution for 1-2 weeks in the refrigerator, then made new dulition from frozen stock - that seemed to work ok for me.
If anyone is interested, puromycin seems to be rather stable in the +4. I`ve aliquoted it 100ug/ml in culture medium about two month ago, and use it from time to time. This week i`ve seed non-resistant cells it to the medium with 1ug/ml of it and almost all of them were dead in two days.
I'm working with puro and hacat cells. Alena, did you have any problems actually getting selection in confluent cultures? I'm wondering if the cells need to be trypsinized to disrupt the epithelial barrier for effective selection. Please let me know if you've gotten this to work.
Hi Philip!
I actually didn't perform a selection on confluent cells - i usually seeded the cells in a complete medium, let them to settle down (from 4 to 12 hours, as it was convenient for me), then changed the medium to the puromycine-containing. i usually seeded them to low rates so the selection would be more active. i think if there are too many cells the resistant ones could metabolize the antibiotic too quickly and it won't have time kill all the non-resistant.
i suppose treating with trypsin could be useful but you may loose more cells, so as some of the resistant ones will die because too much stress.
hei, it s stable at +4 for at least 6 months, and at +37 in the media for at least a week, cheers
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