|Matt Lewis||Department of Pathology |
University of Liverpool
Retroviral infection of primary human keratinocytes (PHKs)
PHK points to bear in mind
PHKs must be actively dividing to be successfully infected
(eg. 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 but I have no feeling for this so I avoid serum altogether and infect in KGM.
Packaging cell points to bear in mind
Packaging cells grow very fast especially PT67s (12hr doubling time)
More than this then defective particles may become a significant factor although at our low titres it is not such an issue.
|Day 0||Resuscitate PHKs into T25s or T75s. |
Include controls for mock infection (T25s or a 6-well plate)
| Day 2|
|Check PHKs, decide that tomorrow will be around 8 cell stage and hence infection day |
ie. You can currently see plenty of 4-cell clumps (if not then wait a day.)Resuscitate packaging cells into DMEM
Resuscitate J2-3T3s into a 6-well plate in DMEM (for titres)
Its best to do a titre for each virus.
|(noon)||Check packaging cells. |
These want to be 50-100% confluent now.
|(pm)|| Refeed packaging cells with KGM |
Not the ideal medium for them but the titre is OK
|Day 3||(Infection day)|
|(am)||Remove supernatants from packaging cells into universals|
Refeed packaging cells with DMEM.
Filter supernatants through 0.45mm filters into fresh universals
If you don't filter then loads of fibroblasts come through and spoil the experiment.
|Refeed PHKs with a 1:1 ratio of infectious supernatant and fresh [KGM + polybrene (12mg/ml)] and put back in the incubator to allow infection to occur for 6-8 hours. |
for T25 use 2ml supernatant + 2ml KGM
|(pm)||Allow infection to proceed all day and refeed with fresh KGM last thing.|
|Day 4||Wait until late on day 4 to begin selection. |
Alternatively, if titres are known to be low, infect again today and begin selection on day 5
Selection of PHKs
G418 only: use 150mg/ml in KGM. Check each day.
|Do not forget to select and kill the mocks|