flashBAC Manual - Oxford Expression Technologies

monolayer is formed, remove the medium, add 0.1 –0.2 mlseed stock virus (diluted to 1-2 ml with medium) and allow thevirus to adsorb for an hour (rock the cells periodically to ensurethe medium covers the cells). After the adsorption period, add10 ml (T75) or 30 ml (T125) medium and allow the virus toreplicate until all the cells are well infected (3-5 days). Removethe medium and store in the dark at 4°C. If setting up multipleflasks, pool the medium before titrating the virus. Titre the virusstock using the protocols in section 8.3. Higher virus titres willalmost always be produced in shake or spinner culture so it iswell worth setting one of the options up in the lab. Shakecultures can be set up very cheaply using similar equipmentdesigned for bacterial cultures.The key to obtaining high virus titres is to use healthy cells inlog phase of culture and to infect cells at a low multiplicity ofinfection (less than 0.1 pfu/cell). It is also important to know thetitre of the recombinant virus stock before setting out on toomuch experimental work to examine gene expression. If thevirus has not amplified for some reason, you need to know this!Infecting cells to monitor gene expressionGene expression can be monitored very simply by infecting 35mm dishes of cells with the recombinant virus at a highmultiplicity of infection (5-10 pfu/cell) to ensure asynchronous infection of every cell. Seed the dishes with cellsto form an even, just sub-confluent monolayer. After allowingthe cells to attach, remove the medium and add the appropriateamount of virus in a volume of 200 µl medium. If you have notyet titrated your virus and do not know the titre, use 200 µl ofundiluted virus. Leave the virus to adsorb at room temperatureOXFORD EXPRESSION TECHNOLOGIES 42