Count cells with the haemocytometer:

number of cells/ 9 squares x 10e4 cells/ml

Cell count at time of splitting should be 1-2 x 10e6 cells/ml

Dilute cells to 1-2 x 10e5/ml, minimum volume for the small flasks is 50 mls

Split cells every 3 (-4) days

Start a large (500 ml minimum) culture at 2x 10e5 cells/ml

Grow cells to 1x 10e6 cells/ ml

Infect cells with virus as determined by titration on 96 well plates

Grow for 2-3 days before harvesting at CONSTANT 27°C

Great care should be taken during harvesting INFECTIOUS medium and cells: use benchcote, beaker with bleach for medium, labcoat, gloves (mask)

Seed 2 x 10e7 cells on 150 cm dishes or in 175 cm2 flask

Let cells adhere for a few hours

Infect cells with virus

Amplification: Use 100-200 µl of viral stock

Titration: 5x 10e4 cells in 100 µl/ well of a 96well plate

Add 100 µl Virus/well,

mix 2x by pipetting carefully up and down

Transfer 100 µl to the next well and repeat, etc

Protein production: Add virus as determined by Titration

DON'T FORGET TO SEAL ALL THE PLATES AND DISHES WITH PARAFILM TO PREVENT DRYING OUT IN THE 27°C INCUBATOR.