Hello. I've been trying to transfect plasmid DNA to my breast cancer cell lines (BT-474 and SK-BR-3) with Thermo Fisher's Lipofectamine 3000 reagent. According to the thermo fisher, those cells have almost %50-70 efficiency but I only managed %10.
I use 25 µL optimem- 1 µL Lipofectamine and 25 µL optimem- 250 ng DNA- 0.5 µL P3000 for BT-474 and
25 µL optimem- 0.5 µL Lipofectamine in one tube and 25 µL optimem- 500ng DNA- 1 µL P3000 for SK-BR-3 as thermo's protocol says. I am using GFP expressing PX458 plasmid to transfect but I don't know why I'm facing this low efficiency problem. Has anyone face that before?
Do you see significant cell death 1-2 days after transfection?
- Perhaps your cells have strict confluency requirements. So if many die after transfection, suddenly you have low confluency and even more die. You could try seeding at higher density the day before transfection
- You could try reducing the amount of lipofectamine/P3000 to reduce cytotoxicity
- ALSO, you can try reducing the amount of DNA you deliver to your cells. I've found that overloading the cells with a ton of plasmid can have cytotoxicity.
It could also be an issue of GFP expression. Is your GFP under a constitutive promoter? Or is it behind another protein sequence that uses a P2A as a separator? Having it behind one or several P2A sequences in succession reduces expression.
What microscope are you using to assess fluorescence? Perhaps your cells are transfected, and simply expressing low GFP (for some reason) If you are using a simple cell culture microscope, you might not be able to pick it up. Try fixing a few wells, doing a DAPI stain, then putting the under cells under a more powerful microscope. Bump exposure time up to 5 seconds and assess if you see any signal.
Also, make sure to change your media the day after transfection.
Thank you for your advice. Gfp is between cas9 and bGH polyA. We don’t have fluorescent microscope in our lab so I never saw gfp expressing cells on microscope, I analyze them on flow cytometry.
Hm flow should pick up a signal as well then..
But in the meantime, is there any way for you to access a fluorescent microscope from another lab?
While you're troubleshooting your protocol, it will be much faster/cheaper to fix and image your cells to assess transfection efficiency, rather than immediately going to flow cytometry.
Unfortunatelly not… I tried fixing cells on cover slips, transfecting cells on chamber slides but everytime I tried, the gfp signal was too low that it was unable to identify.
I am thinking of electroporation. It shows much higher efficiency than lipofectamine I guess…
honestly whatever works, works. Try out electroporation and see what happens.
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