Retroviral supernatants should always be checked for the presence of wildtype retrovirus. Lineage studies can only be properly interpreted if replication-defective retroviruses are used. If replication competent wild-type virus is present, throw away all of the stored supernatant and the batch of retroviral producer cells used and obtain a new lot of cells.
1. On the day before the infection is to be done: Seed a 6-well tissue culture plate with 3T3 fibroblasts at a density of 2 x 104 cells/well.
2. On the day of infection: Infect the 3T3 cells with 2 mL of the test supernatant using 8 |g/mL polybrene (see Note 23). The negative control dish receives fresh 3T3 GM + polybrene.
3. After allowing 3-4 h for absorption, aspirate the media (see Note 24).
4. Refeed the cultures with fresh 3T3 GM + 2 | g/mL polybrene (see Note 25).
5. The day that the 3T3 cells from step 2 become 90% confluent, seed a new 6-well plate with 3T3 fibroblasts at a density of 2 x 104 cells/well for infection the following day. Also, replace the media on the initial set of 3T3 cells infected with test supernatant with half the volume of fresh 3T3 GM.
6. Harvest the supernatant from the 3T3 cells 6-12 h later. Filter the supernatant through a 0.45 |im filter before doing the infection of the second set of 3T3 cells (see Note 26).
7. Infect the second set of 3T3 cells with 2 mL of the filtered supernatant from the original 3T3 cells using 8 | g/mL polybrene. The negative control dish receives fresh 3T3 GM+ polybrene.
8. After allowing 3-4 h for absorption, aspirate media as before.
9. Refeed cultures with fresh 3T3 media + 2 | g/mL polybrene.
10. For testing the presence of a wild-type retrovirus expressing the lacZ gene (see Note 27): Stain with X-gal when the 2nd batch of 3T3 cells become confluent. If the test supernatant contains only replication defective virus, there should be no lacZ-positive cells in the 2nd batch of 3T3 cells.
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