Culture

Organotypic raft culture of primary human keratinocytes.

Retroviral infection of primary human keratinocytes (PHKs) # PHK Key Points PHKs must be actively dividing to be successfully infected. It is important not to allow cells to become confluent as this will prevent trypsinization (even if you do force them all off only 10% will re-attach in the new flask). PHKs must be allowed to 'get going' after resuscitation from freezing, try to infect at the 4–16 cell cluster size (3–4 days?). PHKs differentiate in response to serum and/or Ca++. Apparently they are able to de-differentiate if the exposure was not too long/strong. # Packaging cell points to bear in mind Packaging cells grow very fast especially PT67s (12-hour doubling time). Overconfluence is not so bad (titres remain OK) Half-life of virus is about 4 hours in media at 37ºC. Supernatants are useful at anything from 6 hours to 24 hours. More than this then defective particles may become a significant factor although at our low titres it is not such an issue.

L B Broth