METHODBOOK

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Matt Lewis Department of Pathology
University of Liverpool		 m.lewis@liv.ac.uk

Retroviral infection of primary human keratinocytes (PHKs)

PHK points to bear in mind

PHKs must be actively dividing to be successfully infected
PHKs must never become confluent because they become difficult to trypsinize

(e.g. 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 (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.

Typical protocol

Day 0
Resuscitate PHKs into T25s or T75s.

Include controls for mock infection (T25s or a 6-well plate)

Day 1
Refeed
Day 2
(am)
Check PHKs, decide that tomorrow will be around 8-cell stage and hence infection day

i.e. 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.
If doing lots of different infections then set off the J2-3T3s earlier and trypsinize today into 6-well plates.
They should be 20–80% confluent at infection time.

(noon)
Check packaging cells.

These want to be 50–100% confluent now.
If not then consider (a) resuscitating more and adding on top, or (b) delaying infection until day 4.

(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 0.45µm 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 (12µg/ml)] and put back in the incubator to allow infection to occur for 6–8 hours.

for T25 use 2mL supernatant + 2mL KGM
for T75 use 5mL supernatant + 5mL KGM.
Polybrene is hexadimethrine bromide, Sigma H9268, you want a final conc. of 6µg/mL. Store polybrene stock of 3mg/mL in PBS at 4ºC.

Carry out titration as described (see protocol)
(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 150µg/mL in KGM. Check each day.
Puro only: try 1µg/mL
G418 + Puro: try 50µg/mL G418 + 0.4µg/mL puro.

    Do not forget to select and kill the mocks

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