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Uphoff C.C.,DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures.

Macleod R.A.,DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
Methods in molecular biology (Clifton, N.J.) | Year: 2011

Cancer genes are often deregulated by genomic rearrangements. Accordingly, analysis of the participant chromosomes responsible now occupies a key role in characterizing and identifying cancer cell lines. Cytogenetics may also be used to study the nature and extent of chromosome breakage induced by radiation or chemicals ("clastogenesis"), to distinguish individual cells or clones within a tumor cell population and to monitor the stability of chromosome rearrangements. This chapter describes cytogenetic procedures for characterizing cancer cells in culture. Cell lines allow the use of a wider range of harvesting and hypotonic treatments to optimize metaphase chromosome preparations than that possible with primary cultures. This assists improved banding, fluorescence in situ hybridization (FISH), and Spectral Karyotyping (SKY) analysis for research, rendering cell lines ideal tools for oncogenomics, ideally in parallel with transcriptomic analysis of the same cells. The experience of the writers with more than 800 cell lines has shown that no single hypotonic harvesting protocol is adequate consistently to deliver satisfactory chromosome preparations. Thus, evidence-based protocols are described for hypotonic harvesting, rapid G-banding, and FISH and SKY analysis of cell cultures to allow troubleshooting and fine-tuning to suit the requirements of individual cell lines.

Kampfer P.,Justus Liebig University | Falsen E.,Gothenburg University | Lodders N.,Justus Liebig University | Schumann P.,DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
International Journal of Systematic and Evolutionary Microbiology | Year: 2010

The taxonomic positions of two Gram-positive, endospore-forming rods, strains CCUG 53915T and CCUG 53480T, isolated from an industrial clean-room floor and from a human blood sample, respectively, were studied. 16S rRNA gene sequence similarity studies revealed that both isolates clearly clustered with Sporosarcina species. Strain CCUG 53915T was most closely related to Sporosarcina koreensis and Sporosarcina soli, showing 99.4 and 99.2% 16S rRNA gene sequence similarities to the type strains of these species, respectively. Strain CCUG 53480T showed the highest 16S rRNA gene sequence similarities to the type strains of S. koreensis (98.7%) and Sporosarcina saromensis (98.6%). Strains CCUG 53915T and CCUG 53480T had peptidoglycan type A4α L-Lys-D-Glu. The quinone systems of both strains were composed predominantly of menaquinone MK-7, with small amounts of MK-8. The polar lipid profiles of both strains consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and three unidentified phospholipids. The fatty acid profiles, which comprise anteiso- and iso-branched fatty acids, supported affiliation of the two isolates to the genus Sporosarcina. The results of physiological and biochemical tests and DNA-DNA hybridization data allowed a clear phenotypic and genotypic differentiation of both strains from the most closely related Sporosarcina species. For this reason, it is proposed that strains CCUG 53915T (=DSM 22204T) and CCUG 53480T (=DSM 22203T) represent two novel species in the genus Sporosarcina, with the names Sporosarcina contaminans sp. nov. and Sporosarcina thermotolerans sp. nov., respectively. © 2010 IUMS.

Drexler H.G.,DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The advent of continuous human leukemia-lymphoma cell lines as a rich resource of abundant, accessible, and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first leukemia-lymphoma cell lines were established in 1963 and since then large numbers of new cell lines have been described. The major advantages of continuous leukemia-lymphoma cell lines are the unlimited supply and worldwide availability of identical cell material and the infinite viable storability in liquid nitrogen. These cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and by specific genetic alterations. Here some of the more promising techniques for establishing new leukemia-lymphoma cell lines and the basic principles for culturing these cells are described. Several clinical and cell culture parameters might have some influence on the success rate, e.g., choice of culture medium and culture conditions, specimen site of the primary cells, and status of the patient at the time of sample collection.

Dirks W.G.,DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
Methods in molecular biology (Clifton, N.J.) | Year: 2011

The main prerequisition for any research, development, or production programs involving cell lines is whether a cell line is authentic or not. Microsatellites in the human genome harboring short tandem repeat (STR) DNA markers allow the identification of individual cell lines at the DNA level. Polymerase chain reaction (PCR) amplification of eight highly polymorphic microsatellite STR loci and gender determination have been proven to be the best tools for screening the uniqueness of DNA profiles in an STR database. The main Biological Resource Centers (BRCs), ATCC, DSMZ, JCRB, and RIKEN, have generated large databases of STR cell line profiles for identity control. In cooperation with the Japanese BRCs, DSMZ has piloted the generation of the most comprehensive international reference database, which is linked to a simple search engine for interrogating STR cell line profiles. The tool of online -verification of cell line identities is available on the respective homepages of JCRB and DSMZ ( http://cellbank.nibio.go.jp/cellbank_e.html , http://www.dsmz.de/STRanalysis ). The following sections describe a rapid, practical, inexpensive, and reliable method available to students, technicians, and scientists.

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