Hello dear researchers, i am generating a stable cell line of HEK293T cells using lentivirus, i want to ask about the puromycin kill curve,if i seed my cells in a 12-well plate on day 1, Q1) when should i start adding different concentrations of puromycin....Q2) while adding puromycin do i need to remove all of the old media from wells or i should remove half of it and add new puro containing media......one thing more whenever i start adding puro containing media the cells starts detaching from well slightly, even with adding it very gently...
i used random concentrations of puro(2 ug/ml,4ug/ml, and 10ug/ml) but the control cells are not dying after 24 hours.
If you seed cells on Day 1, normally you would wait overnight/24 hours to allow the cells to adhere to the plate properly and start growing again. This should also help with cells detaching during addition of media + puromycin. When adding puromycin, it is better to completely replace the media i.e. add the puromycin to the media, mix well, then place on cells. This also avoids having slightly different puromycin concentrations depending on how much of the original volume of media is lost due to evaporation etc. Puromycin is usually left on for 48 hours, but there are some cells that require a longer time. Some people even continue to culture their cells with puromycin. You can also try higher concentrations.
Dear Dr. Aakif Inayat
You need to first perform the puromycin kill curve before the actual experiment to detect the minimum antibiotic concentration to use that kills 100% of untreated control cells in 2–15 days from the start of antibiotic selection.
You may use the following protocol.
1. You plate HEK293T cells at an optimum cell density and culture overnight at 37 degree C and 5% CO2.
2. The next day replace complete growth medium with the selection medium supplemented with a range of puromycin concentrations. Include untreated control cells with growth medium only (no puromycin added). You may use antibiotic working concentration in the range of 0.5–10 µg/mL for puromycin.
3. Monitor the cells daily using a microscope and observe the percentage of surviving cells.
4. Approximately every 2–3 days replace medium with freshly prepared selection medium containing the range of antibiotic concentrations which you are testing.
5. Optimum effectiveness for puromycin would be reached in 2-7 days.
Q1) when should I start adding different concentrations of puromycin?
After 24 hours when HEK293T cells have attached to the substratum.
Q2) While adding puromycin do I need to remove all of the old media from wells, or I should remove half of it and add new puro containing media?
You should remove the entire old media (complete media) and add the selection medium supplemented with a range of puromycin concentrations.
Q3. Whenever I start adding puro containing media the cells start detaching from well slightly, even with adding it very gently?
HEK293T cells are not so adherent. They tend to loosely attach to the substratum.
Best.
agree with Malcolm Nobre you need to determine the puromycin dose for your cells empirically. you want them to die over a longer timeframe. expecting death in 24 hours means the dose is too high. check the literature and do 3 doses above and below. yes it is tedious but necessary and will save time and headache in the long run
Thank you very much research gate community for your valuable suggestions and answers,it helps me a lot during my research journey.i started puromycin kill curve,hope I can find the suitable concentration....
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