A new method for the enrichment of single renal proximal tubular cells and their first use in the comet assay.
A protocol was developed to isolate and enrich single renal proximal tubular cells, performing the following steps: in situ kidney perfusion; isolation of renal tissue pieces by collagenase digestion; selective enrichment of proximal tubular fragments by Percoll gradient centrifugation; and isolation of single proximal tubular cells by digestion of proximal tubular fragments with trypsin. The mean enrichment rate, determined by the glucose-6-phosphatase staining method, was 78.9% with a mean cell viability of 93.8%. After modification of the comet assay protocol, genotoxicity in proximal tubular cells could be investigated. A dose-dependent genotoxic effect of ethyl methanesulphonate in these cells was proven.
Brendler-Schwaab
S Y
SY
Department of Carcinogenicity and Genotoxicity, Bayer AG, Wuppertal, Germany.
Herbold
B A
BA
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification; D004249:DNA Damage; D004586:Electrophoresis / Q000379:methods; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley
D009153:Mutagens; D004247:DNA; D005020:Ethyl Methanesulfonate