Titration of retrovirus

Points to bear in mind

Target cells have to be dividing to be infected.

The viral RNA/protein complex cannot enter the nucleus and has to wait for the nuclear membrane to dissolve at mitosis.

Typical protocol

The day before infection set up J2-3T3s in 6-well plates

We use J2-3T3s because they give nice discreet colonies after selection
They want to be 20-80% confluent on the day of infection
Some retrovirus cannot be titred on rodent cells such as 3T3. For example, packaging lines using the gibbon ape leukaemia virus (GALV) env protein must be titred on human cells.

On infection day;

Do the main infection first, then keep back at least 200ml of filtered supernatant for the titration.
For each titration set up 6 Eppendorf tubes each containing 800ml of DMEM, labelled 1-6.
Prepare 6ml of [DMEM + 9mg/ml polybrene] in a universal

Eg. if doing 3 titrations then prepare 18ml (say 20ml)
Polybrene is hexadimethrine bromide, Sigma H9268, you want a final conc. of 6mg/ml
Store polybrene stock of 3mg/ml in PBS at 4C.

Add the 200ml of supernatant to Eppendorf no.1 and mix by inverting several times
Transfer 200ml from Eppendorf 1 to Eppendorf 2 and mix by inverting.
Continue through all 6 tubes.
Refeed the J2-3T3s with 1ml/well of [DMEM + 9mg/ml polybrene]
Add 0.5ml from each Eppendorf to each of the wells 1-6.

Printed protocols recommend a 10-fold dilution series. This is like car speedos going to 165mph.
We have never seen any colonies in the last well using a 5-fold dilution series.

Leave cells to infect until late next day, then start selection.

Selection of J2-3T3s

G418; use 600mg/ml in DMEM,it takes around a week to get the result.

Puromycin; use 12mg/ml in DMEM, it can take several days to get the result.

I don't count colonies down the microscope.- I remove the media and let the plate dry overnight. Then I score the colonies visually.