Interleukin-1 alpha is released during transfection of keratinocytes.
Keratinocytes are known to produce, store, and release IL-1 alpha and therefore we suspected that the DNA-mediated cell transfection procedure may release the stored IL-1 alpha from keratinocytes into the medium. Using enzyme-linked immunosorbent assay, we determined the IL-1 alpha concentration in culture supernatants during keratinocyte transfection. The following transfection methods were compared: lipofection with lipofectACE and lipofectAMINE (GIBCO), Ca3(PO4)2 co-precipitation, and polybrene-dimethylsulfoxide (DMSO). The supernatants were collected immediately prior to transfection, after 5-h incubation with lipofectin or Ca3(PO4)2, and 24 and 48 h after transfection. In the polybrene-DMSO method, the supernatant was also collected immediately before and after DMSO shock. LipofectAMINE caused the highest release of IL-1 alpha, whereas the lipofectACE and polybrene-DMSO mediated transfection with confluent cells released the least. The other two methods released intermediate levels of IL-1 alpha. Our data indicate that a substantial amount of IL-1 alpha is released during the keratinocyte transfection procedure, which can affect the results of transfection in studies of gene expression.
Komine
M
M
Ronald O. Perelman Department of Dermatology, NYU Medical Center, New York 10016.
Freedberg
I M
IM
Blumenberg
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D002411:Cation Exchange Resins / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007231:Infant, Newborn; D007375:Interleukin-1 / Q000378:metabolism*; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008055:Lipids / Q000494:pharmacology; D008297:Male; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D002411:Cation Exchange Resins; D007202:Indicators and Reagents; D007375:Interleukin-1; D008055:Lipids; C086724:Lipofectamine; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D004121:Dimethyl Sulfoxide