A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages.
Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.
Mack
K D
KD
Medicine and the AIDS Program, University of California San Francisco and San Francisco General Hospital, 94110, USA.
Wei
R
R
Elbagarri
A
A
Abbey
N
N
McGrath
M S
MS
D000328:Adult; D001358:Avian Sarcoma Viruses; D002448:Cell Adhesion; D002478:Cells, Cultured; D002738:Chloroquine; D003470:Culture Media; D003587:Cytomegalovirus / Q000235:genetics; D003911:Dextrans; D004121:Dimethyl Sulfoxide; D004305:Dose-Response Relationship, Drug; D004983:Ethanolamines; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D005990:Glycerol; D016325:HIV Long Terminal Repeat; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D003470:Culture Media; D003911:Dextrans; D004983:Ethanolamines; D002738:Chloroquine; D008156:Luciferases; D005990:Glycerol; C007369:2-diethylaminoethanol; D004121:Dimethyl Sulfoxide