Help on transfecting MCF7 and HCT 116 cells - (Oct/22/2008 )

Hi all,

Does anyone have the efficient protocol to transfect HCT116 and MCF7 cells using lipofectamine 2k? I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent.

I did several transfections in HCT116 using Lipofectamine 2000 and it worked perfectly in our experimental conditions. I am planning to use the same reagent in MCF7 cells (in recent future), so I don't have solid experience in this cell line yet.

I have used this reagent to transfect luciferase plasmids. Let me outline my brief protocol (24-well plate design);

Day 1 (Seeding):
-cell seeding at a density of 100,000 cells/well (cell should be 70-80% confluent on the day of transfection)

Day 2 (Transfection):
i) Change fresh OptiMed 450 ul without PBS washing.
ii) Transfection complex
# Tube 1- 25 ul OptiMem and add 0.6 ug of plasmid DNA (firefly construct) and 0.05 ug pRL-TK (renilla control) ---then incubate 5 min at RT
# Tube 2- 25 ul OptiMem and add 1.2 ul of Lipofectamine-- then incubate 5 min at RT
# Add the content of tube 1 (diluted DNA) to tube 2 (lipofectamine) and mix gently by pipetting (2-3 times)
# Further incubate 20-25 min at RT
iii) After incubation, add 50 ul dropwise directly above the cells (not to the wall of the well-plate) the tranfection complex.
iv) Incubate 16 h (Although 3-5 h is enough since transfection process is rapid, overnight incubation is OK).

Day 3 (Recovary and drug treatment):
i) Change fresh medium containing full supplement (10% FBS) and incubte 6-8 h to recovary.
ii) After recovary, wash cells once with warm HBSS and flood serum free media (I did drug treatment for 24 h in serum free condition).

Day 4 (Lysis)
i) Wash once with HBSS
ii) Add 100 ul passive lysis buffer
iii) Freeze at -70 for 4 h or overnight

Day 5 (Luciferase)
i) Dual luciferase (Promega)

Hope these help to design your own experiment.

Good luck!!
Thapa