Prior to viral collection, transfected cells should be checked by immunostaining with the 3C2 monoclonal anti-GAG antibody (18) to ensure that the retrovirus has spread throughout the culture (see Subheading 3.6.). To optimize retroviral titers, it is important that the supernatant is collected from newly confluent or near-confluent dishes of dividing cells. Viral titers can be enhanced by minimizing both the volume and serum concentration of the media used for collection. Collection of supernatant containing infectious viral particles using reduced serum CEF media facilitates resuspension of the viral particles after concentration by centrifugation and does not adversely affect viral titer.
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