Retrovirus titration

Contributed by Matt Lewis <hop2kin@yahoo.co.uk>

Titration of retrovirus lines.

Note, target cells must be dividing to be infected. As the viral RNA/protein complex cannot enter the nucleus entry must wait for nuclear membrane to dissolve at mitosis.

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

J2-3T3s are recommended as they provide discreet colonies after selection

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.

• Check cells are 20-80% confluent on the day of infection
• Prepare 6ml/titration of [DMEM + 9ug/ml polybrene] in a 50ml universal tube e.g. for 3 titrations prepare 18ml.

Polybrene is hexadimethrine bromide (Sigma H9268). Final concentration should be 6ug/ml. Store polybrene stock of 3mg/ml in PBS at 4C.

• For each titration set up 6 Eppendorf tubes each containing 800ul of DMEM, labelled 1-6.
• Add the 200ul of supernatant to Eppendorf no.1 and mix by inverting several times
• Transfer 200ul from Eppendorf 1 to Eppendorf 2 and mix by inverting.
• Continue through all 6 tubes producing a serial dilution.
• Refeed the J2-3T3s with 1ml/well of [DMEM + 9ug/ml polybrene]
• Add 0.5ml from each Eppendorf to each of the wells 1-6.
• Incubate cells overnight at 37’C to start infection. Select the following day.
• For G418; use 600ug/ml in DMEM, it takes around a week to get the result.

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

To avoid counting colonies down the microscope remove media and let the plate dry overnight. You can then score colonies visually.

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