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Braunschweig, Germany

MacLeod R.A.F.,DSMZ German Collection of Microorganisms and Cell Cultures | Drexler H.G.,German Biological Resource Center
Methods in Molecular Biology | Year: 2013

Cytogenetic analysis is performed on cell cultures for several reasons, notably, to perform identity checks by verifying species of origin or the retention of key chromosome rearrangements in cell lines described previously. De novo chromosome analysis is usually performed when characterizing cancer cell lines for the presence of neoplastic rearrangements associated with specific tumors. This usually involves fluorescence in situ hybridization (FISH) using clones covering gene loci near recurrent chromosome breakpoints. Chromosome breakage is an important endpoint in radiation biology and mutagenesis, enabling cell lines to be used for measuring genotoxic dosage and repair. Finally, cytogenetic analysis may be performed to monitor stability in culture. Unlike most preparative techniques, chromosome preparation resists standardization. Hence, procedures must be optimized for each cell line. Thus, evidence-based protocols are described for hypotonic harvesting, rapid G-banding, FISH, and Spectral Karyotyping (SKY) analysis of cell cultures to allow troubleshooting and fine-tuning to suit the requirements of individual cell lines. © 2013 Springer Science+Business Media, LLC. Source


Uphoff C.C.,DSMZ | Drexler H.G.,German Biological Resource Center
Methods in Molecular Biology | Year: 2013

Mycoplasma contaminations have a multitude of effects on the cultured cell lines that may influence the results of experiments or pollute bioactive substances used in human medicine. The elimination of mycoplasma contaminations of cell cultures has become a practical alternative to discarding and reestablishing important or irreplaceable cell lines. Different quinolones, tetracyclines, and pleuromutilins shown to have strong antimycoplasma properties are employed for the decontamination. We provide detailed protocols to assure eradication of mycoplasma, to prevent formation of resistant mycoplasma strains, and to cure heavily contaminated and damaged cells. To date, we have not detected any consistent and permanent alterations to eukaryotic cells either during or after the treatment. © 2013 Springer Science+Business Media, LLC. Source


Uphoff C.C.,DSMZ | Drexler H.G.,German Biological Resource Center
Methods in Molecular Biology | Year: 2013

Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory and can also be used to determine the contaminating species. The described assay can be performed in less than 3 hours, including sample preparation, DNA extraction, PCR run, and analysis of the PCR products. Special emphasis is given to steps taken to avoid false-negative results due to the presence of inhibitors of the Taq polymerase in the crude samples and the interpretation of the results. The technique can also be adapted to the requirements of the pharmacopoeia. © 2013 Springer Science+Business Media, LLC. Source


Dirks W.G.,German Biological Resource Center | Drexler H.G.,German Biological Resource Center
Methods in Molecular Biology | Year: 2013

Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem repeat (STR) DNA markers allow individualization of cell lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of human cell lines and represents an international reference technique. The major cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a human cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent cells within a human cell culture population. Since authentic cell lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of human cell lines of interest. © 2013 Springer Science+Business Media, LLC. Source

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