flashBAC Manual - Oxford Expression Technologies

2 Using aseptic technique, add 0.5 ml (no more) of therecombinant virus seed stock (from 8.1) to the cellculture and incubate with shaking or stirring (asappropriate) until the cells are well infected (normally 4-5days).• Virus-infected cells become uniformly rounded andenlarged, with distinct enlarged nuclei. They appeargrainy when compared with healthy cells under thephase-contrast inverted microscope.• Virus-infected cells have an increased need foroxygen and therefore the contents of the shake flasksshould be shaken at quite high speeds to maximizeaeration. The surface area to volume ratio shouldalso be as large as possible for maximum gasexchange – do not overfill flasks!3 When the cells appear well infected with virus, harvestthe culture medium by centrifugation at 3000 rpm, at4°C for 15 minutes. Decant aseptically and store therecombinant virus in the dark at 4°C.• The virus inoculum may be stored for 6-12 months orlonger in the dark at 4°C. The titre of the virus willstart to fall after a time and after storage for morethan 3-4 months it is recommended to titre the virusbefore using it – it may require re-amplification. Thetitre seems to drop more quickly in serum-freemedium and the addition of serum to 2-5% may helpto prevent this.• Virus may also be frozen at –80°C for a longer periodof time. If frozen, avoid multiple freeze and thawOXFORD EXPRESSION TECHNOLOGIES 28