Retroviral infection of PHKs

Contributed by Matt Lewis <>

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.

  • Resuscitate frozen PHKs into T25s or T75s in suitable media. Remember to include controls for mock infection (T25s or a 6-well plate)
  • Incubate overnight at 37’C
  • Refeed cells with appropriate media.
  • Incubate overnight at 37’C
  • Check PHKs and determine if you can currently see plenty of 4-cell clumps - if so tomorrow will reach 8-cell stage (infection day). If not, 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. *

  • Check packaging cells. These should be 50–100% confluent now. If not then consider (a) resuscitating more and adding on top, or (b) delaying infection until day 4.
  • Refeed packaging cells with KGM. Not the ideal medium for them but the titre is OK
  • Incubate all cells overnight; tomorrow is infection day.
  • Remove supernatants from packaging cells (put to one side) into universals and refeed 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.*

  • Allow infection to proceed all day.
  • Refeed with fresh KGM.
  • Incubate overnight to day 4 to begin selection.
  • Selection:

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.

This method is based, with permission, on an original protocol available here.