Detecting Mycoplasma Contamination in Cell Cultures by Polymerase Chain Reaction

Uphoff, Cord C.; Drexler, Hans G.

doi:10.1007/978-1-61779-080-5_8

Cited by 138 publications

(111 citation statements)

References 3 publications

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“…Therefore, interpretation of the biological tests on these cells will be affected by incorrect results. In order to increase the quality and safety of the biological products prepared in animal cell cultures, US Food and Drug Administration (FDA) strongly recommends the application of mycoplasmafree cells approved by proper detection tests (Cheng et al 2007;Dabrazhynetskaya et al 2011;Huang et al 2008;Uphoff and Drexler 2011). It has been reported (in different studies) that between 5 and 87 % of the cell lines have been contaminated by mycoplasmas.…”

Section: Introductionmentioning

confidence: 99%

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

et al. 2014

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Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert Ò and molecular. Enzymatic evaluation was performed using the mycoalert Ò kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert Ò method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (\20 min).

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Section: Introductionmentioning

confidence: 99%

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

et al. 2014

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“…Cell lines in the maximal range of up to 20 passages were used for the present study. All cell lines tested negative for mycoplasma contamination by polymer chain reaction (PCR) methods (16). Cell lines were authenticated using the short tandem repeats (STR) testing.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of microRNA-1323 restores sensitivity to radiation by suppression of PRKDC activity in radiation-resistant lung cancer cells

Han

et al. 2015

Oncology Reports

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Abstract.Resistance to radiation is a major problem in cancer treatment. The mechanisms of radioresistance remain poorly understood; however, mounting evidence supports a role for microRNAs (miRNAs) in the modulation of key cellular pathways mediating the response to radiation. The present study aimed to identify specific miRNAs and their effect on radioresistant cells. The global miRNA profile of an established radioresistant lung cancer cell line and the corresponding control cells was determined.

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“…A maximum of 20 passages were used. To avoid mycoplasma contamination, the cell line was assessed monthly using polymerase chain reaction (PCR) (23). For PCR, the ATCC Universal Mycoplasma Detection Kit (ATCC) was used.…”

Section: Methodsmentioning

confidence: 99%

DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair

Peng

et al. 2012

Molecular Medicine Reports

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Abstract. The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated γ-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following γ-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent γ-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced γ-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of γ-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the quantity of γ-H2AX determines the radioresistance of cells. The DNA repair pathway is important in mediating radioresistance, and treatment with the DNA-PKcs inhibitor, NU7026 restored the acquired radiation resistance.

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Detecting Mycoplasma Contamination in Cell Cultures by Polymerase Chain Reaction

Cited by 138 publications

References 3 publications

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Knockdown of microRNA-1323 restores sensitivity to radiation by suppression of PRKDC activity in radiation-resistant lung cancer cells

DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair

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