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LookOut Mycoplasma Elimination Kit - ADVERTISEMENT

Paige L. Dobrovolny1

1Product Management, Sigma-Aldrich

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    Abstract

    The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In addition, since mycoplasma have no cell wall and are therefore resistant to many antibiotics. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.

    The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.

    Current methods for the inactivation or elimination of mycoplasma in cell cultures are antibiotic-based. Antibiotic therapies do not always result in successful elimination of contaminants. Moreover, antibiotics exhibit cytotoxic properties in that they are capable of modifying the metabolism of eukaryotic cells and they promote the development of resistant mycoplasma. The LookOut Mycoplasma Elimination Kit is gentle on cells with minimal cytotoxicity usually less than 20% and removes mycoplasma contamination in three passages.

    The main ingredient integrates selectively into the mycoplasma membrane to disrupt it through a biophysical process. Given this unique mechanism of action mycoplasma are not likely to become resistant. This ingredient not been shown to cause any changes in normal cell characteristics. In short the LookOut Mycoplasma Elimination Kit is the method of choice for the elimination of mycoplasma contamination in cultured cells.

    Protocol

    LookOut Mycoplasma Elimnation Kit Bulletin

    1. Cell Preparation and Treatment

    1. Select the appropriate media for the contaminated cells and prepare as necessary, noting that FBS concentration should be 5% for the initial elimination step. Place 4.5 ml into a cell culture flask or petri dish.
    2. Warm the media in the flask to 37 °C.
    3. Add the contents (500 μl) of the Mycolplasma Elimination - Initial Treatment (product number M4569) to the warmed media and gently mix. Figure 1 highlights the entire workflow of the Lookout Mycoplasma Elimination Kit.
    4. Split and passage the cells as appropriate for the contaminated cell line as thinly as possible, given the nature of the cell line.
    5. Add 5 ml of the cell suspension to the media/Elimination Reagent mixture prepared.
    6. View the flask under a microscope once the cells and media mixture have been combined to ensure single cell suspensions, separate cell aggregates as necessary.
    7. Add enough media to bring the cells to the correct concentration, noting that FBS concentration should be 5%, and grow cells to 80-90% confluent.
    8. Passage the cells at the usual rate. Add the contents of one vial (500 μl) of Mycoplasma Elimination Reagent - Final Treatment (product number M4444) to 9.5 ml of passaged cells in fresh medium. Resume routine FBS concentration. Repeat this through two additional passages.

    2. Representative Results

    After the completion of the final treatments your cells should be mycoplasma free. Confirmation of whether or not the mycoplasma has been eliminated can be confirmed by using the LookOut Mycoplasma Detection Kit. Representative results using the LookOut Mycoplasma Detection Kit can be seen in Figure 2.

    Figure 1
    Figure 1.

    Figure 2
    Figure 2. Relevant Amplicon Bands.

    References

    1. Zinöcker, S., Wang, M.-Y., Gaustad, P., Kvalheim, G., Rolstad, B., et al. Mycoplasma Contamination Revisited: Mesenchymal Stromal Cells Harboring Mycoplasma hyorhinis Potently Inhibit Lymphocyte Proliferation In Vitro. PLoS ONE 6 (1), e16005, doi:10.1371/journal.pone.0016005 (2011).
    2. Deutschmann, S.M., Kavermann, H., & Knack, Y. Validation of a NAT-based Mycoplasma Assay According to European Pharmacopiea. Biologicals: Journal of the International Association of Biological Standardization. 38, 238-248 (2010).
    3. Eterpi, M., McDonnell, G., & Thomas, V. Decontamination Efficacy Against Mycoplasma. Letters in Applied Microbiology. 52, 150-155 (2010).
    4. Drexler, H.G. & Uphof, C.C. Mycoplasma Contamination of Cell Cultures: Incidence, Sources, Effects, Detection, Elimination, Prevention. Cytotechnology. 39, 75-90 (2002).
    5. Hopert, A., Uphoff, C.C., Wirth, M., Hauser, H., & Drexler, H.G. Specificity and Sensitivity of Polymerase Chain Reaction (PCR) in Comparison with Other Methods for the Detection of Mycoplasma Contamination in Cell Lines. Journal of Immunological Methods. 164, 91-100 (1993).
    6. Kong, H., Volokhov, D.V., George, J., Ikonomi, P., Chandler, D., Anderson, C., & Chizhikov, V. Amplification of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT). Applied Microbiology and Biotechnology. 77, 223-232 (2007).
    7. Kotani, H., Butler, G., Heggan, D., & McGarrity, G.J. Elimination of Mycoplasmas from Cell Cultures by Novel Soft Agar Technique. In Vitro Cellular & Developmental Biology: Journal of the Tissue Culture Association. 27A, 509-513 (1991).
    8. Mattson, J.G. & Johansson, K.E. Oligonucleotide Probes Complementary to 16S rRNA for Rapid Detection of Mycoplasma Contamination in Cell Cultures. FEMS Microbiology Letters. 107, 139-144 (1993).
    9. Volokhov, D.V., Kong, H., George, J., Anderson, C., & Chizhikov, V. Biological Enrichment of Micoplasma Agents by Cocultivation with permissive Cell Cultures. Applied and Environmental Microbiology. 74, 5383-5391 (2008).
    10. Volokhov, D.V., Graham, L.J., Borson, K.A., & Chizhikov, V. Mycoplasma Testing of Cell Substrates and Biologics: Review of Alternative Non-Microbiological Techniques. Molecular and Cellular Probes., (2011).
    11. Zhi, Y., Mayhew, A., Seng, N., & Takle, G.B. Validation of PCR method for the Detection of Mycoplasmas According to the European Pharmacopoeia Section 2.6.7. Biologicals. 38, 232-237 (2010).

    Discussion

    The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. It is for this reason that the LookOut Mycoplasma Elimination kit was developed to quickly and efficiently eliminate mycoplasma.

    Media used to maintain and pass cells differs with each cell line. When using the LookOut Mycoplasma Elimination kit it is important to remember to continue to use the same culture medium and subculture routine (with the exception of the FBS concentration). The activity of the initial treatment can be affected by the concentration of lipids and proteins in the reaction mixture, therefore the FBS concentration should not be greater than 5%.

    When viewing the cell suspension under the microscope take care to ensure a single cell suspension, if there are any aggregates of cells they should be separate. This will help ensure proper treatment of each cell.

    In addition to using the LookOut Mycoplasma Elimination kit is it always important to remember to use proper aseptic techniques and maintain a clean working environment to decrease the chances of mycoplasma contamination.

    Disclosures

    Author is an employee of Sigma-Aldrich, who is the funding source of this video.

    Acknowledgements

    Author is an employee of Sigma-Aldrich, who is the funding source of this video.

    Materials

    Name Company Catalog Number Comments
    LookOut DNA Erase Sigma- Aldrich L8917 Spray for surface cleaning
    LookOut DNA Erase refill Sigma-Aldrich L9042 Refills for L8917
    LookOut Mycoplasma Detection Kit Sigma-Aldrich MP0035
    Mycoplasma Stain Kit Sigma-Aldrich MYC1

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