Titering Virus-Protocol

Plate 2x10⁶ cells in Lux plates in total of 3ml (e.g. 1ml Sf9 at 2x10⁶ and 2ml medium)

Allow to attach, then aspirate supernatant:

For each virus make a 10^-5, 10^-6, 10^-7 dilution in Grace's complete

e.g. make 1:100 dilution (5ml in 5 ml), then

dilute 1:100 (50ml in 5ml)à10^-5

dilute 1:10 (500ml in 5ml)à10^-6

dilute 1:10 (500ml in 5ml)à10^-7

Pipet 1ml of each dilution onto a separate dish of cells.

Incubate for 1h at 28⁰C, agitating by rocking every 10 min, then aspirate virus. Meanwhile, pre-warm 40ml medium to 37⁰C. Melt 5% LMP-agarose, mix well. Allow to cool to app, 28.37⁰C, then carefully pipet 4ml per dish onto cells. Incubate at 28⁰C for 5-6 days.

On day 5 or 6 make 1% LPM-agarose/grace's as before, but add to it 1ml 1% Trypman blue, mix and add 2ml to each plate.

In 1 or 2 days, count plaques.