DEAE-DEXTRAN PROTOCOL FOR TRANSIENT TRANSFECTION OF COS-7 CELLS AND HARVEST OF CONDITIONED MEDIUM

1.     Plate COS-7 cells the day prior to the transfection.  On the day of transfection the cells should be 50-80% confluent.

2.     Warm 1X PBS and DEAE-Dextran solutions to 37°C.

3.     For a 75 cm²  flask:   using a sterile tube, dilute 10 µg of DNA to a final volume of 540 µl in 1X PBS.  Add 28 µl of DEAE-Dextran (10 mg/ml solution) and mix by gently tapping the tube.

4.     Remove culture medium from the cells.  Wash the cells twice with 1X PBS (2 x 10 ml per flask).

5.     Add the DNA/DEAE-Dextran mixture and spread it evenly over the cells. 

6.     Incubate the plates at 37°C for 30 minutes.  Occassionally rotate/rock the plates to ensure the cells do not dry.

7.     Gently add 6 ml of growth medium (DMEM with supplements + 10% FBS) per flask, followed by an addition of 60 µl of chloroquine (8 mM solution in 1X PBS) per flask.  Incubate between 2.5 and 4 hrs at 37°C.  Monitor the cells for evidence of cytotoxicity. At the end of the incubation or when cytotoxicity is evident then gently change the medium and replace with Phenol Red Free DMEM with supplements + 10% FBS (10-12 ml/flask).

8.     After an additional 24 h incubation, wash the cells with 1X PBS (10 ml/flask) and replace with Phenol Red and Serum Free DMEM with supplements (10-12 ml/flask).

9.     Collect conditioned medium and store at –20 to –40°C (do not use a refrigerator/freezer for storage)

10.  Purify using Nickel (recommended buffer pH 7.8-8.0) and Anti-Flag columns.