I transfected BxPC3 cells with 5µg Akt plasmid using Lipofectamine 2000. I also had a control where I added only Lipofectamine 2000. Surprisingly, I observed 60-70% cell death in the wells transfected with Akt plasmid whereas no cell death was observed in the wells treated with lipofectamine 2000 alone. Below is the protocol I followed for transfection:
1. Seed 0.2 x 10 6 cells per well in a 6 well plate
2. Incubate overnight
3. Aspirate the media and wash with 1X PBS
4. Add 1.7ml of OPTI-MEM reduced serum media
5. Incubate at 37 o c for 45 mins
6. In the meantime, quantitate the plasmid and the plasmid concentration was 1182.2 ng/µl
I had 3 wells to transfect with 5µg Akt plamid and to avoid the losses I took lipofectamine 2000 and plasmid for 4 wellss
7. Mix 600µl OPTI-MEM + 36µl lipofectamine 2000
8. Mix 600µl OTI-MEM + 20µg Akt plasmid.
9. Incubate for 5 minutes at room temp.
10. Add 600µl of diluted DNA to 600µl LPF
11. Incubate for 8 mins at room temp
12. Add 300µl DBA-lipid complexes drop by drop per well
13. Incubate at 37 o C for 8 hours
14. Replace the OPTI-MEM reduced serum free media with fresh RPMI media with 10% FBS
15. Incuabte at 37 o C for 48 hours
Hence, each well contains 9µl Lipofectamine 2000 and 5µg plasmid
and I treated one well only with 9µl Lipofectamine 200 where no cell death was observed.
Any suggestions would be of a great help to me. Please assist in this situation.
Pancreatic cancer cell lines are known to be a little bit picky. It looks like your plasmid is resulting the cell death (given your controls), and I would look at previous literature with the plasmid to see what kind of effects you are expecting from it. Your plasmid concentration also seems a bit high, like really high. Here's a transfection kit designed for BxPC-3 (https://altogen.com/product/bxpc-3-transfection-reagent-pancreatic-cancer-cells/) and the protocol there says to use around 750 ng plasmid per 45 microliters of other reagents, which is magnitudes lower than your concentration. I would try using a lower concentration and see if that gives you better results.
Thank you so much for your efforts Dr Bremmer . In the past we have used BxPC3 cells for transfection but I will try using a lower concentratin of Akt plasmid (1µg) for transfection. I am trying to overexpress Akt and please suggest if 1µg Akt plasmid will be sufficient. Ted Bremmer
I would suggest trying a range of concentrations (if your resources allow). I'm not sure what you mean by 1µg of Akt plasmid - is it per well or with the 600 µl of medium? In either case, I haven't used the Akt plasmid before, so I don't know what the optimum concentration will be. We always have a pre-run when culturing populations of stably-transfected cells to determine the optimum concentration, and the protocol for the transfection kit I mentioned in my earlier response says to mix 750 ng of plasmid per 45 µl of other reagents, so that might be a concentration to try out as well.
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