Detection of Mycoplasma in cell cultures

Young, Lesley; Sung, Julia; Stacey, Glyn; Masters, John R

doi:10.1038/nprot.2010.43

Cited by 437Microbial infections and disease researchViral Infectious Diseases and Gene Expression in InsectsBacterial Identification and Susceptibility Testing

Detection of Mycoplasma in cell cultures

Young, Lesley, Sung, Julia, Stacey, Glyn, Masters, John R·2010·Nature Protocols

DOI 10.1038/nprot.2010.43

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Abstract

Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3-4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.

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Contamination with mycoplasma was ruled out by PCR testing according to standard protocols.
“Contamination with mycoplasma was ruled out by PCR testing according to standard protocols37.”
From Common germline variants of the human APOE gene modulate melanoma progression and survival (2020) · cites this paper as [37]

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Background
Extracellular vesicles and their cells of origin: Open issues in autoimmune diseases
Haghighitalab, A., Dominici, M., Matin, M. M., Shekari, F., Warkiani, M. E., Lim, R., Ahmadiankia, N., Mirahmadi, M., Bahrami, A. R., & Bidkhori, H. R. (2023) · Frontiers in Immunology · doi:10.3389/fimmu.2023.1090416 · Cited by 20 · see citing works
Aging-induced isoDGR-modified fibronectin activates monocytic and endothelial cells to promote atherosclerosis
Park, J. E., JebaMercy, G., Pazhanchamy, K., Guo, X., Ngan, S. C., Liou, K. C. K., Lynn, S. E., Ng, S., Meng, W., Lim, S., et al. (2021) · medRxiv · doi:10.1101/2021.03.02.21252419 · Cited by 20
NRF2 Dysregulation in Hepatocellular Carcinoma and Ischemia: A Cohort Study and Laboratory Investigation.
Ziv, E., Zhang, Y.-R., Kelly, L., Nikolovski, I., Boas, F., Erinjeri, J., Cai, L., Petre, E., Brody, L., Covey, A., et al. (2020) · Radiology · doi:10.1148/radiol.2020200201 · Cited by 20 · see citing works
“All cells were used at less than 10 passages and tested bimonthly for mycoplasma contamination (22).”
Background
Integrin α3β1 on Tumor Keratinocytes Is Essential to Maintain Tumor Growth and Promotes a Tumor-Supportive Keratinocyte Secretome
Longmate, W. M., Varney, S. D., Power, D., Miskin, R. P., Anderson, K. E., DeFreest, L. A., Water, L. V. D., & DiPersio, C. M. (2020) · Journal of Investigative Dermatology · doi:10.1016/j.jid.2020.05.080 · Cited by 20 · see citing works
“MK lines are tested several times per year for Mycoplasma using a PCR-based method (Young et al., 2010), and all studies were conducted within 6 months of latest test date (12–2018).”
Method
The quantitative impact of 3′UTRs on gene expression
West, J. D., Smith, H. J., Vu, L. T., Fogarty, E. A., Matreyek, K. A., Fowler, D. M., & Grimson, A. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkaf568 · Cited by 17 · see citing works
“Cell lines were routinely verified to be free of mycoplasma contamination using a previously described PCR protocol with primers 5 (cid:2) TGC ACC ATCTGTC ACTCTGTTAACCTC and 5 (cid:2) GGGA GCAAA CA GGA TT AGA T ACCCT [ 50 ].”
Excitable Rho dynamics control cell shape and motility by sequentially activating ERM proteins and actomyosin contractility
Marshall-Burghardt, S., Migueles-Ramírez, R. A., Lin, Q., El Baba, N., Saada, R., Umar, M., Mavalwala, K., & Hayer, A. (2024) · Science Advances · doi:10.1126/sciadv.adn6858 · Cited by 23 · see citing works
Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness
Lin, C., Levy, P. L., Alflen, A., Apriamashvili, G., Ligtenberg, M. A., Vredevoogd, D. W., Bleijerveld, O. B., Alkan, F., Malka, Y., Hoekman, L., et al. (2024) · Cancer Cell · doi:10.1016/j.ccell.2024.02.016 · Cited by 21 · see citing works
Treating Cells as Reagents to Design Reproducible Assays
Riss, T., Moravec, R. A., Duellman, S. J., & Niles, A. L. (2021) · SLAS DISCOVERY · doi:10.1177/24725552211039754 · Cited by 26 · see citing works
Molecular docking prediction and in vitro studies elucidate anti-inflammatory effect of Garcinia extract against inducible nitric oxide synthase and cyclooxygenase-2 targets
Kalita, A., Das, M., Das, B., & Baro, M. R. (2022) · Beni-Suef University Journal of Basic and Applied Sciences · doi:10.1186/s43088-022-00214-2 · Cited by 23 · see citing works
“The cells were tested for any kind of Mycoplasma contamination before the experiments following the PCR-based method [34, 35].”
Evaluation of an aldo-keto reductase gene signature with prognostic significance in colon cancer via activation of epithelial to mesenchymal transition and the p70S6K pathway.
Canlı, S. D., Seza, E., Sheraj, I., Gomceli, I., Turhan, N., Carberry, S., Prehn, J., Gure, A., & Banerjee, S. (2020) · Carcinogenesis · doi:10.1093/carcin/bgaa072 · Cited by 18 · see citing works
Metabolomics reveals mycoplasma contamination interferes with the metabolism of PANC-1 cells
Yu, T., Wang, Y., Zhang, H., Johnson, C. H., Jiang, Y., Li, X., Wu, Z., Liu, T., Krausz, K. W., Yu, A., et al. (2016) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-016-9525-9 · Cited by 18 · see citing works
“Contamination of cell cultures with mycoplasma presents a challenging issue in terms of detectability and prevention since no overt changes are observed in media pH, turbidity, and direct microscopic visualization [3, 4, 7].”
Background
MicroRNA miR-331-3p suppresses osteosarcoma progression via the Bcl-2/Bax and Wnt/β-Catenin signaling pathways and the epithelial-mesenchymal transition by targeting N-acetylglucosaminyltransferase I (MGAT1)
Bi, W., Yang, M.-Y., Xing, P., & Huang, T. (2022) · Bioengineered · doi:10.1080/21655979.2022.2083855 · Cited by 17
“Monthly mycoplasma tests to ensure cells purity [25].”
Retracted
The Role of miR-375-3p and miR-200b-3p in Gastrointestinal Stromal Tumors
Gyvytė, U., Lukoševičius, R., Inčiūraitė, R., Streleckiene, G., Gudoityte, G., Bekampytė, J., Valentini, S., Salteniene, V., Ruzgys, P., Šatkauskas, S., et al. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21145151 · Cited by 17
“Cell lines were tested for mycoplasma contamination using specific primers [43].”
Method
Rapid Detection of Mycoplasma-Infected Cells by an ssDNA Aptamer Probe.
Liu, Y.-T., Jiang, W.-Q., Yang, S., Hu, J., Lu, H.-B., Han, W., Wen, J.-G., Zeng, Z.-H., Qi, J., Xu, L., et al. (2019) · ACS Sensors · doi:10.1021/acssensors.9b00582 · Cited by 17
Mitoribosomal synthetic lethality overcomes multidrug resistance in MYC-driven neuroblastoma
Borankova, K., Krchniaková, M., Leck, L. Y. W., Kubistova, A., Neradil, J., Jansson, P. J., Hogarty, M. D., & Skoda, J. (2023) · Cell Death and Disease · doi:10.1038/s41419-023-06278-x · Cited by 16 · see citing works
“Subsequently, cell con ﬂ uency was determined every 4 h using live cell imaging system Incucyte ® SX1 (Sartorius, Göttingen, Germany) and plotted relative to initial values.”
A low-cost simple test for weekly detection of Mycoplasma hyorhinis and arginini contaminations in cell cultures and viral preparations.
Lai, M., Iacono, E., Spezia, P. G., Lottini, G., Rocca, V. L., Quaranta, P., Pistello, M., & Freer, G. (2021) · Journal of Virological Methods · doi:10.1016/j.jviromet.2021.114327 · Cited by 16
Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture
Degeling, M. H., Maguire, C. A., Bovenberg, M. S. S., & Tannous, B. A. (2012) · Analytical Chemistry · doi:10.1021/ac2033112 · Cited by 19 · see citing works
Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer
Kandala, S., Ramos, M., von Voithenberg, L. V., Diaz-Jimenez, A., Chocarro, S., Keding, J., Brors, B., Imbusch, C. D., & Sotillo, R. (2023) · Cell Reports · doi:10.1016/j.celrep.2023.113266 · Cited by 21 · see citing works
The Cytoskeleton Effectors Rho-Kinase (ROCK) and Mammalian Diaphanous-Related (mDia) Formin Have Dynamic Roles in Tumor Microtube Formation in Invasive Glioblastoma Cells
Becker, K. N., Pettee, K. M., Sugrue, A., Reinard, K. A., Schroeder, J. L., & Eisenmann, K. M. (2022) · Cells · doi:10.3390/cells11091559 · Cited by 16 · see citing works
“All cell lines were routinely screened for mycoplasma, as described [41].”
Method
CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition
Musicant, A. M., Parag‐Sharma, K., Gong, W., Sengupta, M., Chatterjee, A., Henry, E. C., Tsai, Y., Hayward, M. C., Sheth, S. H., Betancourt, R., et al. (2021) · Cell Reports · doi:10.1016/j.celrep.2021.108768 · Cited by 19 · see citing works
“All cell lines were confirmed mycoplasma-free by PCR as previously described (Young et al., 2010) and using mycoplasma detection primers (see Table S6).”
PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway
Hodgins, J. J., Abou‐Hamad, J., O’Dwyer, C., Hagerman, A., Yakubovich, E., de Souza, C. T., Marotel, M., Buchler, A., Fadel, S., Park, M. M., et al. (2024) · The Journal of Experimental Medicine · doi:10.1084/jem.20221721 · Cited by 18 · see citing works
Concurrent inhibition of ALK and SRC kinases disrupts the ALK lung tumor cell proteome
Diaz-Jimenez, A., Ramos, M., Helm, B., Chocarro, S., Frey, D. L., Agrawal, S., Somogyi, K., Klingmüller, U., Lu, J., & Sotillo, R. (2024) · Drug Resistance Updates · doi:10.1016/j.drup.2024.101081 · Cited by 13 · see citing works
Tick Activity, Host Range, and Tick-Borne Pathogen Prevalence in Mountain Habitats of the Western Carpathians, Poland
Zając, Z., Kulisz, J., Woźniak, A., Bartosik, K., Foucault-Simonin, A., Moutailler, S., & Cabezas-Cruz, A. (2023) · Pathogens · doi:10.3390/pathogens12091186 · Cited by 14 · see citing works
“In the real-time PCR reaction, we applied primers identical to those used in a previously published paper [25,26].”
Influential
Manassantin A inhibits tumour growth under hypoxia through the activation of chaperone-mediated autophagy by modulating Hsp90 activity
Byun, J., Lee, S.-H., Moon, E. J., Park, M.-H., Jang, H., Weitzel, D., Kim, H.-H., Basnet, N., Kwon, D., Lee, C.-T., et al. (2023) · British Journal of Cancer · doi:10.1038/s41416-023-02148-7 · Cited by 14 · see citing works
“All cell lines were routinely tested for mycoplasma contamination using a PCR-based method [13].”
Method
TSC2 regulates tumor susceptibility to TRAIL‐mediated T‐cell killing by orchestrating mTOR signaling
Lin, C., Traets, J. J. H., Vredevoogd, D. W., Visser, N. L., & Peeper, D. S. (2023) · The EMBO Journal · doi:10.15252/embj.2022111614 · Cited by 18 · see citing works
Critical role of PD-L1 expression on non-tumor cells rather than on tumor cells for effective anti-PD-L1 immunotherapy in a transplantable mouse hematopoietic tumor model
Rodríguez-Barbosa, J.-I., Azuma, M., Zelinskyy, G., Pérez‐Simón, J. A., & del Rio, M. (2020) · Cancer Immunology Immunotherapy · doi:10.1007/s00262-020-02520-z · Cited by 19 · see citing works
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
Kazemiha, V. M., Amanzadeh, A., Memarnejadian, A., Azari, S., Shokrgozar, M., Mahdian, R., & Bonakdar, S. (2014) · Cytotechnology (Dordrecht) · doi:10.1007/s10616-013-9640-9 · Cited by 14 · see citing works
“This explains the advantageous of the PCR over the classical methods especially in the case of low level contaminations (right before invasion of infection or after eradication of agents) (Young et al. 2010).”
Background
Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination
Dowdell, A. S., Cartwright, I. M., Kitzenberg, D., Kostelecky, R. E., Mahjoob, O., Saeedi, B. J., Welch, N., Glover, L. E., & Colgan, S. P. (2022) · Cell Reports · doi:10.1016/j.celrep.2022.111409 · Cited by 14 · see citing works
“All cell lines used were tested for mycoplasma at least once a month using established protocols (Young et al., 2010).”
Method
An in vitro selective inhibitory effect of silver(I) aminoacidates against bacteria and intestinal cell lines and elucidation of the mechanism of action by means of DNA binding properties, DNA cleavage and cell cycle arrest.
Rendošová, M., Gyepes, R., Maruščáková, I. C., Mudroňová, D., Sabolová, D., Kello, M., Vilková, M., Almáši, M., Huntošová, V., Zemek, O., et al. (2020) · Dalton Transactions · doi:10.1039/d0dt03332d · Cited by 13
15‐LOX‐1 has diverse roles in the resensitization of resistant cancer cell lines to doxorubicin
Kazan, H., Urfali-Mamatoglu, C., Yalcin, G. D., Bulut, O., Sezer, A., Banerjee, S., & Gunduz, U. (2020) · Journal of Cellular Physiology · doi:10.1002/jcp.29375 · Cited by 13
Multi-primer qPCR assay capable of highly efficient and specific detection of the vast majority of all known Mycoplasma
Salling, H. K., & Bang-Christensen, S. R. (2016) · Biologicals · doi:10.1016/j.biologicals.2016.03.003 · Cited by 12 · see citing works
Keratinocyte integrin α3β1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism
Longmate, W., Norton, E., Duarte, G., Wu, L., DiPersio, M. R., Lamar, J. M., & DiPersio, C. (2024) · Matrix Biology · doi:10.1016/j.matbio.2024.02.003 · Cited by 12 · see citing works
Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas
Rideau, F., Villa, A., Belzanne, P., Verdier, E., Hosy, E., & Arfi, Y. (2022) · bioRxiv · doi:10.1128/spectrum.00645-22 · Cited by 12 · see citing works
“As mycoplasmas are frequent contaminants of these cultures (79), mycoplasmologists might be barred from accessing these facilities with live cells.”
Background
Downregulation of ARID1B, a tumor suppressor in the WNT subgroup medulloblastoma, activates multiple oncogenic signaling pathways
Deogharkar, A., Singh, S. V., Bharambe, H. S., Paul, R., Moiyadi, A. V., Goel, A. H., Shetty, P. M., Epari, S., Gupta, T., Jalali, R., et al. (2021) · Human Molecular Genetics · doi:10.1093/hmg/ddab134 · Cited by 12 · see citing works

Authors: Young, L., Sung, J., Stacey, G., & Masters, J. R.
Affiliations: University College London (GB)
Corresponding author: Masters, John R
ORCID: Stacey, Glyn
Venue: Nature Protocols
Publisher: Springer Science and Business Media LLC
Published: 2010-04-22
Volume: 5
Issue: 5
Pages: 929-934
ISSN: 1750-2799 (ISSN-L)
1754-2189
DOI: 10.1038/nprot.2010.43
OpenAlex: W2092105616
Cited by: 437
References: 11
Citations by year: 2026: 23 · 2025: 44 · 2024: 43 · 2023: 53 · 2022: 32 · 2021: 41 · 2020: 36 · 2019: 40 · 2018: 35 · 2017: 19 · 2016: 12 · 2015: 17 · 2014: 14 · 2013: 9 · 2012: 10
FWCI: 8.25
Type: journal article
Language: en
Open access: closed
Licence: www.springer.com/tdm (tdm)
Locations: Nature Protocols (publishedVersion)
PubMed (publishedVersion)
UCL Discovery (University College London) (submittedVersion)
Topics: Life Sciences › Immunology and Microbiology › Microbiology › Microbial infections and disease research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Bacterial Identification and Susceptibility Testing
Keywords: mycoplasma detection, cell culture contamination, PCR detection, laboratory quality control, tissue culture
MeSH terms: Bacteriological Techniques, DNA, Bacterial, Electrophoresis, Agar Gel, Mycoplasma, Sensitivity and Specificity, Staining and Labeling, Time Factors, Polymerase Chain Reaction

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Detection of Mycoplasma in cell cultures

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