|Matt Lewis||Department of Pathology |
University of Liverpool
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.
The day before infection set up J2-3T3s in 6-well plates
We use J2-3T3s because they give nice discreet colonies after selection
On infection day;
Do the main infection first, then keep back at least 200ml of filtered supernatant for the titration.
Eg. if doing 3 titrations then prepare 18ml (say 20ml)
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.
Selection of J2-3T3s
G418; use 600mg/ml in DMEM, it takes around a week to get the result.
I donít count colonies down the microscope.† I remove the media and let the plate dry overnight. Then I score the colonies visually.