A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts.
BACKGROUND
The Host Cell Reactivation Assay (HCRA) is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances.A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products) and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing).


RESULTS
In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP) and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis.The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts.


CONCLUSIONS
The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity of different cell types can be compared with each other. The described assay is also highly flexible, and the activity of other repair mechanisms can be determined using modifications of this method.
Burger
Katharina
K
Department of Biomedical Engineering, University of Applied Sciences, Anton-Günther-Strasse 51, 72488 Sigmaringen, Germany.
Matt
Katja
K
Kieser
Nicole
N
Gebhard
Daniel
D
Bergemann
Jörg
J
D002478:Cells, Cultured; D004260:DNA Repair; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008544:Melanocytes / Q000378:metabolism*; D010957:Plasmids; D014162:Transfection
D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins