Collection of Viral Supernatant

1. Split transfected CEF's 1:5 from newly confluent plates into an appropriate number of 10-cm tissue-culture dishes. Incubate cells until they are 80-90% confluent (1 or 2 d).

2. Discard medium and rinse once with sterile PBS.

3. Replace with 5 mL of reduced serum CEF media containing 5% FCS and 1% CS. Do not include pen/strep or polybrene in the culture media. Incubate cells overnight.

4. Collect supernatant and put into sterile 50 mL plastic centrifuge tubes. Supernatant can be stored at -70°C at this point.

5. If the monolayer of cells is still intact, collect a second aliquot of supernatant by placing an additional 5 mL of reduced-serum CEF media on plates and incubating for a further 4-24 h (see Note 7).

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