Wittrup: HEK293-F Transfection

PEI

1. Dissolve 50 mg PEI (Polysciences 23966) in 50 mL ddH20. Heat at 55C for at least 30 minutes until dissolved and clear. 2. Adjust pH to 7 with HCl. Filter sterilize, aliquot, and store at -80C.

HEK293-F 1 L Transfection Protocol for IgG

1. Prepare 500 ug light chain DNA and 500 ug heavy chain DNA (a concentration of 1 mg/mL in water or buffer EB). 2. Grow cells to a density of 1.4 – 1.6 x 10^6 cells per mL (logarithmic growth phase) in two 1 L flasks, 360 mL media volume each. (Subculture cells about every two days to 0.2 x 10^6, do not grow to above 2 x 10^6 cells per mL, do not dilute below 0.1 x 10^6). 3. Thaw 1 mg/mL PEI from -80 at room temp or 37C. 4. Allow optipro to come to room temperature. 5. Add DNA to 19 mL optipro. Mix gently. 6. Add 2 mL PEI to 18 mL optipro. Mix gently. 7. Add DNA solution to PEI solution, mix gently. Incubate 5-10 minutes at room temperature. 8. Set up two 2 L bottles, each with 500 mL 1 x 10^6 cells/mL in Freestyle Media. 9. Slowly add 20 mL DNA:PEI solution to each bottle. 10. Place bottles in roller incubator at 37C and incubate 6-8 days.