MycoAlert

Mycoplasmas are one of the most common contaminants present in cell culture laboratories. Their presence is usually anything but welcome, and unfortunately often goes unnoticed. Read in this blog article how MycoAlert® can be used to identify and consequently eliminate a mycoplasma contamination.

Dr Ann-Cathrin Volz Dr Ann-Cathrin Volz (10)

Mycoplasma contamination

Mycoplasma are simple prokaryotes and measure only 0.2-0.8 µm1. Since they rely on hosts as source for many of their essential nutrients, they are usually attached to the external surface of cells 2. Mycoplasma lack a proper cell wall (fig. 1) and are therefore not susceptible to classic antibiotics3.

Fig. 1: Schematical structure of mycoplasma.Mycoplasma contaminations often go undetected for months, as the organisms are not visible in the microscope and do not cause visible changes in turbidity or pH. While staying off the grid, mycoplasma can substantially affect your cells. Although they grow slowly and won’t kill the cells outright, they still inhibit cellular metabolism and growth and cause DNA fragmentation, disrupt nucleic acid synthesis, and increase sensitivity to apoptotic inducers4,5,6,7. Consequently, these effects can seriously impact the reliability, consistency, and reproducibility of any experimental result of your cell-based assays. Regular testing for mycoplasma contamination should therefore be routinely performed in your cell culture laboratory for quality control.
The MycoAlert Assay Kits from Lonza, offer a sensitive and easy way to implement testing for mycoplasma contaminations in the routine quality control.

MycoAlert principle

Lonza’s MycoAlert Mycoplasma Detection kits exploit the activity of mycoplasmal enzymes. These enzymes are found in the vast majority of mycoplasma species but are not present at all in eukaryotic cells. If mycoplasma expressing such enzymes are present in a sample, they convert the MycoAlert assay substrate to generate ATP (fig. 2). The level of converted ATP can then be determined with a luminescence-based reaction. For this purpose, luciferase is included in the MycoAlert mycoplasma detection kit. This enzyme uses ATP to convert its substrate luciferin to oxyluciferin which leads to the generation of a luminescent light signal. The produced luminescent signal can then be measured with a luminometer or a luminescence microplate reader.

Since the targeted mycoplasmal enzymes occur in 44 species, the MycoAlert assay can be used for the detection of a broad spectrum of mycoplasma contaminations.
Fig. 2: Schematic depiction of the conversion of MycoAlert Substrate to ATP through mycoplasmal enzymes, and the subsequential conversion of ATP and luciferin to oxyluciferin, AMP and a luminescent signal.

The MycoAlert assay can be used to detect mycoplasma contaminations in various samples such as fresh media, supplements or water. The assay is started, by simply addingthe MycoAlert Reagent to a sample. Lysing substances in the MycoAlert Reagent lead to the lysis of viable mycoplasma cells and the release of mycoplasmal enzymes (fig. 3). Since the MycoAlert Reagent already includes the luciferase enzyme, any background ATP present in the sample will be used up in an initial bioluminescent reaction. Accordingly, the first read (Read A) is done to provide information about the level of the innate ATP in the sample, which shouldn’t lead to substantial luminescence counts. High initial counts would indicate a contamination with ATP. After the addition of the MycoAlert Substrate, mycoplasmal enzymes, if present, generate new ATP. Subsequently, the added luciferase uses the produced ATP to generate oxyluciferin and light from luciferin. In this step, the produced luminescent signal is proportional to the available ATP specifically derived from mycoplasmal enzymes in the sample.Fig. 3: MycoAlert assay workflow. The luminescent signal of Read B (after the addition of MycoAlert Substrate) is divided by Read A (background ATP), resulting in a ratio indicative of the presence or absence of mycoplasma.

 

This potential luminescence signal increase is detected with Read B. Calculating the ratio of Read B/Read A allows to evaluate the presence or absence of mycoplasma in a sample. If no mycoplasma are present, the second reading shows no increase over the first and therefore results in a small ratio (<0.9 with the MycoAlert Assay and <1.0 with MycoAlert PLUS). If mycoplasmal enzymes exceeding the kits detection limit are found in a sample, Read B exceeds Read A leading to a ratio >1.2.
 
Lonza’s MycoAlert mycoplasma detection kits are available in two versions, the MycoAlert® Mycoplasma Detection Kit and the MycoAlert® PLUS Mycoplasma Detection Kit. Principle and workflow of both kits are the same - they mainly differ in their sensitivity. The MycoAlert PLUS Detection Kit leads to an increased light production already with minimal amounts of present mycoplasmal enzymes. It is therefore more suitable for the detection of weak contaminations, or contaminations in an early state. It is also suitable for mycoplasma detection with less sensitive luminometers.

Fig. 4: MycoAlert signal curve over time. Signal for the MycoAlert reaction in the presence of a MycoAlert positive control sample is shown in blue, and a mycoplasma negative control in red.

 

MycoAlert measurement on BMG LABTECH microplate readers

The detection of a mycoplasma contamination with the MycoAlert kits requires the availability of a (microplate) reader capable of measuring luminescence. BMG LABTECH’s microplate readers LUMIstar Omega, FLUOstar Omega, VANTAstar, CLARIOstar Plus and PHERAstar FSX are all perfectly suited for the detection of a mycoplasma-related increase of luminescence using the MycoAlert kits. All of BMG LABTECH’s luminescence microplate readers exhibit a sensitivity exceeding the requirements specified by Lonza for both kits. To simplify the process and minimise possible errors during the manual steps, it is recommended to use BMG LABTECH's reagent injectors for adding the substrate and reagent into the microplate. This way, the generated luminescence signal can be monitored in a single protocol, including the reagent addition as well as read A and Read B measurements. Beyond that, BMG LABTECH’s MARS Data Analysis Software offers effortless evaluation of the MycoAlert assay signal ratio and easy interpretation of results.

If you are interested in more details about the detection of MycoAlert on BMG LABTECH plate readers, please see our AppNotes on the topics: Lonza’s MycoAlert assay on a BMG LABTECH plate reader and Detecting mycoplasma cell culture contamination using the CLARIOstar microplate reader.

MycoAlert Assays enable the sensitive and rapid detection of mycoplasma contaminations – especially in combination with BMG LABTECH microplate readers, which significantly simplify the assay workflow, and data acquisition, as well as the analysis and the evaluation of the assay results.

MycoAlert is a registered trademark of Lonza.

References

  1. Hay, R. J., Macy, M. L. & Chen, T. R. Mycoplasma infection of cultured cells. Nature 339, 487–488 (1989).
  2. Uphoff, C. C. & Drexler, H. G. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. Vitr. Cell. Dev. Biol. - Anim. 38, 79–85 (2002).
  3. Geraghty, R. J. et al. Guidelines for the use of cell lines in biomedical research. Br. J. Cancer 111, 1021–1046 (2014).
  4. Miller, C. J. et al. Mycoplasma infection significantly alters microarray gene expression profiles. Biotechniques 35, 812–814 (2003).
  5. McGarrity, G. J., Vanaman, V. & Sarama, J. Cytogenetic effects of mycoplasmal infection of cell cultures: A review. In Vitro 20, 1–18 (1984).
  6. McGarrity, G. J. & Kotani, H. Cell Culture mycoplasmas. in The Mycoplasmas 353–390 (1985). doi:10.1023/A:1022913015916.
  7. Drexler, H. G. & Uphoff, C. C. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39, 75–90 (2002).
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