DMEM+10% DMEM with high glucose
Pen/strep solution, fungizone is optional
10% BCS (I use Hyclone #2151)

DMEM- DMEM without any additives (no antibiotics or serum)

DMEM+2% DMEM with high glucose
Pen/strep solution, fungizone is optional
2% BCS (I use Hyclone #2151)
*You can substitute serum-free medium such as Gibco's Opti-MEM for DMEM+2%**

Lipofection reagent

Cos-7 cells, available from ATCC

Sterile DNA, about 10µg is easy to precipitate

Allow cells to grow overnight

Solution A: 1µg DNA in 100µl DMEM-

Solution B: 10µl Lipofectin in 100µl DMEM-

Mix A+B, incubate 15minutes at room temperature
Meanwhile, wash cells with 2ml DMEM- to remove any trace of serum or antibiotics

Add 0.8µl of DMEM- to A+B

Very gently add A+B to cells

Very gently place cells into incubator, grow overnight

Next morning gently remove lipofectin and gently give cells 2ml DMEM+2% or Opti-MEM

Allow cells to grow for 2-5 days then analyze for protein production

I usually run mini-gels with 5 to 20µl of conditioned medium, which is enough to see bands on a western-blot if you have a good antibody

Using low-serum medium or serum-free medium increases the concentration of your protein relative to the total protein in the conditioned medium

I usually include three controls
1. Cos-7 cell CM
2. Cos-7 cell CM lipofected without vector
3. Cos-7 cell CM lipofected with vector but no insert