Day 3

The media should be changed, and if appropriate, the cells should be split. The cells should be passaged as usual, making sure they are split at least every other day. For all viral subtypes, except A, 2 |g/mL of polybrene should be included in the media at this stage to enhance infection. Viral supernatant should be collected (see Subheading 3.4.) 7-10 d following transfection. Similarly, transfected cells can be used for grafting experiments (see Subheading 3.5.) after 7-10 d in culture (see Note 6).

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