Titering Virus-Protocol
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 (5(l in 5 ml), then
dilute 1:100 (50(l in 5ml)(10-5
dilute 1:10 (500(l in 5ml)(10-6
dilute 1:10 (500(l in 5ml)(10-7
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.
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