What are the advantages/disadvantages of using fibronectin vs. polybrene?
A while back fibronectin was used to increase transduction/infection of hematopoietic stem cells and other hard to transduce primary cells. They would first coat the plate with fibronectin, add virus to the plate which will bind to the fibronectin, then plate the cells on top. Polybrene works by countering the electrostatic charges between the virus and cell membrane, which both have a negative charge on its surface. Polybrene is a cation. Polybrene is toxic to some cell lines and primary cells, whereas fibronectin probably is not. For primary cells, there is no standard transduction protocol because primary cells differ greatly in the way the culture conditions can be manipulated. Fibronectin has been used to increase transduction/infection of hematopoietic stem cells and other hard to transduce primary cells. First coat the plate with fibronectin, add virus to the plate which will bind to the fibronectin, then plate the cells on top. Fibronectin is non toxic for most cells. Another option to consider is adding polybrene or DEAE-dextran to the virus stock prior to infection. This is used to increase transduction efficiency by counterbalancing the charge-charge interactions between the virus particle and the surface of the cell. However, cationic reagents such as these are very toxic to primary cells in culture and should not be used. A reduction in serum concentration in the culture can also facilitate transduction, but some primary cells do not handle a reduction in serum over the time frame of infection. To avoid these problems, generally one can reduce the volume of the culture media to where it just covers the cells and add straight virus directly onto the culture media. Let the infection proceed for 6-8 hours with gentle rocking every hour or so, and then add the normal culture media to the cells. This way there is little perturbation to the cells. The amount of virus added needs to be determined empirically for each cell type. We recommend setting up three infections with multiplicity of infections equal to 5, 10, and 50. Not all cell types transduce equally well.