1. Quick thaw the cells in 37 C water bath and add to 10 ml of growth medium. Spin 5 min at 800 rpm to remove DMSO from freezing medium. DMSO is toxic to growing cells. Resuspend in 10 ml growth medium in 75 cm2 TC flask and place in 5% CO2/37 C incubator.
2. Plate approximately 5 x 105 cells into 60mm petri dishes. Wait until the cells reach 80% confluent.
3. Dilute 2-4 ug DNA of each construct to be transfected into 250 ul of OptiMEM or DMEM medium. Add 8 ul of Plus Reagent (Invitrogen # 11514-015) to the DNA/MEM mix. Let sit 15 min at room temperature.
4. Separately, mix 12 ul of Lipofectamine (Invitrogen # 18324-111) with 250 ul of OptiMEM or DMEM.
5. Mix DNA/MEM/Plus solution with Lipofectamine/MEM. Let sit 15 min at room temperature.
6. Remove growth medium from 60mm cell cultures. Rinse once with 2 ml OptiMEM or DMEM, replace with another 2 ml MEM to remove serum from the plate.
7. Add preincubated DNA/Plus/Lipofectamine/MEM solution to the cells. Mix gently.
8. Grow cells 3 hr – overnight. Add 2.5 ml N2a medium and continue culture.
If developing stable transfectants, add G418 (Gibco/Life Tech # 1181-031) to the growth medium to select for positive cells. Use at 400-800 ug/ml for selection of clones, and 200-400 ug/ml for maintenance.
9. 48 hours post transfection, harvest the cells for the future assay.
DMEM, high glucose, w/L-gluatmine Gibco # 11965-092 250ml
Fetal bovine serum, qualified Gibco # 26140-079 50ml
OptiMEM 1 medium Gibco # 31985-070 250ml
Penicillin-Streptomycin Gibco # 15140-122 5ml
Filter to 0.2 um. Stable for several months at 4 C.