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{Why is Polybrene added to a transduction?}
Polybrene (also called Hexadimethrine bromide) is a cationic agent that is thought to shield the negative surface charges of virus and cell, thus increasing transduction rates by decreasing repulsion. Depending on the cell type it may have a huge effect and can increase the titer 2-fold up to 10-fold. The standard concentration is 8 µg/ml in the cell culture medium during transduction (stock solution: 8 mg/ml in water or PBS). Polybrene is toxic to the cells in higher concentrations and some delicate primary cells may not like it at all. Lower concentrations of about 4 µg/mL may already show the maximal effect and if necessary the optimal concentration can be determined experimentally. Also Protamine sulfate and other cations have been shown effective. Read more: PubMed-link.

{Why is Chloroquine added during the transfection?}
Chloroquine shall decrease the intracellular degradation of plasmid DNA in the lysosomes, thus increasing the amount of plasmid DNA that reaches the nucleus and gets expressed. The standard concentration is 25 µM in the cell culture medium during transfection (stock solution: 25 mM in PBS). But I also had good titers once I forgot to add Chloroquine, and I haven't ever quantified its effect. The literature says something like "great effect on some cells, no effect on other cells". Read more: PubMed-link.

{Where do I get the packaging plasmids from?}
Packaging plasmids are available e.g. at www.AddGene.org (klick here to open their list).
We use third generation packaging (pMDLg/pRRE + pRSV-Rev), but packaging should work with any second or third generation HIV-1 packaging system. For example, I have tested psPAX2 second generation packaging with comparable results. You always need another plasmid expressing the envelope protein, like pMD2.G or phCMV-VSV-G.

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