Abstract:
:This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Hsu CY,Uludağ Hdoi
10.1038/nprot.2012.038subject
Has Abstractpub_date
2012-04-19 00:00:00pages
935-45issue
5eissn
1754-2189issn
1750-2799pii
nprot.2012.038journal_volume
7pub_type
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