Is it possible to use Lipofectamine to transfect plasmid DNA into MCF-7 cells using a 96 well method? Is there a specific protocol you could recommend?
This question is part of the following Ask The Expert session:
Improve lentiviral production using Lipofectamine 3000 reagent
Company: Thermo Fisher Scientific, Inc.
Job Title: R&D Scientist
Yes, Lipofectamine 3000 transfection reagent works well for delivery DNA into MCF-7 cells.
We get ~60% transfection efficiency (GFP plasmid) by using Lipofectamine 3000 to transfect MCF-7 cells. Here are some suggestions for 96-well plate format:
• The day before transfection, seed 25,000 MCF-7 cells per well of a 96-well plate. This translates to ~85% confluence at the time transfection
• Next Morning, transfect the cells (for each transfection reaction):
a) Tube 1: Dilute 100ng DNA ( high quality, endotoxin-free) + 0.2ul of P3000 reagent in 5ul Opti-MEM I
b) Tube 2: Dilute 0.3ul Lipofectamine 3000 reagent in 5ul Opti-MEM I
c) Combine the two tubes and incubate at room temperature for 5 minutes to form transfection complex
d) Following the complex formation step, add 10ul complex directly to the cell culture medium in each well.
e) 6 hours post transfection, change cell media and replace with fresh MCF-7 culture medium
f) Post-tfx 48hrs, harvest the cells
The medium change at 6 hours post transfection is not required when using Lipofectamine 3000, but if the cells are sensitive, such as the case with MCF-7, medium changing will help to reduce the cell death.
ATCC MCF-7 cells growth rate is low. We use Gibco DMEM, high glucose, GlutaMax Supplement, pyruvate + 10%FBS to culture the MCF-7. The supplement sodium pyruvate will help MCF-7 growth a lot.