transfection of macrophages

Ian Cassady I.Cassady at mailbox.uq.oz.au
Tue Dec 12 20:23:42 EST 1995


Hi,

We have spent some time optimizing conditions for transient transfection 
of murine macrophage cell lines including J774.  The chemical methods 
are not appropriate because these cells are very pinocytotic so all they 
tend to do is concentrate the reagents until they become toxic.  We 
found electroporation to be the only efficient method and use the 
following conditions: 5,000,000 healthy cells in 250 ul of 
RPMI1640+10%FCS; 4mm gap cuvette; usually 10ug plasmid DNA; Zap at 
250->300 V, 960 uFaraday at room temperature on BioRad GenePulser with 
Capacitance Extender; time constant usually 40-50 milliseconds; add 
medium to cells immediately after zap; incubate a minimum of 12 hours.

We usually use RAW264 as these transfect really well, J774 work less 
well. Primary macrophages die. Have a look at the following papers.

Cassady et al NAR 19:6839-6847 (1991)
Stacey et al Immunol Cell Biol 71:75-85 (1993)

Good luck,
Ian Cassady





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