Most experiments that rely on lentiviral technology to deliver genetic elements benefit greatly by starting with pure high-titer virus. With some easy-to-transduce cell systems grown in routine medium, such as DMEM, it may be possible to squeak by with a dilute viral supernatant containing proteins and factors from the packaging cell culture. However, protocols involving cells that require different media (esp. cells that grow in suspension) or cells that are difficult-to-transduce (stem cells, blood cells, etc.) will likely require viral concentrations 100-fold more dense than just the supernatant. Freezing small, highly-concentrated aliquots is much more efficient also.
The problem, of course, has always been how to concentrate the virus without concentrating the exogeneous proteins and other factors present in the supernatant. The traditional approach usually involves either an inconvenient and time-consuming sucrose-gradient ultracentrifugation or a messy PEG precipitation followed by centrifugation which co-precipitates most of the proteins and other impurities in the cells and media.
Fortunately, our experience over the years manipulating lentiviruses has lead to a solution — LentiFuge™ Viral Concentration Reagent. It aggregates viral particles together so they can be isolated and separated from other impurities in the medium by standard high-speed centrifugation. The whole LentiFuge isolation protocol takes just 2 hours. One hour incubation with the Reagent, and a second hour for centrifugation in a Beckman JA-14, JA-10, or similar rotor. After these two short steps, the virus is simply resuspended and either used immediately or aliquoted and frozen.
Data from a recent experiment (below) shows that LentiFuge not only concentrates the virus by 2-fold, but it also removes impurities from the viral preparation. The figure shows the transduction efficiency ratio of concentrated virus versus pre-concentrated virus. With LentiFuge, essentially all the virus was recovered and the LentiFuge Reagent gave much better yields than the more traditional PEG protocol.
Viral supernatant was collected at 48-hrs post-transfection concentrated using either 1) high-speed centrifugation, 2) LentiFuge treatment with centrifugation, 3) PEG (polyethylene glycol) treatment with centrifugation, or 4) not concentrated. The resulting concentrated virus was used to transduce cells in culture.
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