Introduction

Cell cultures are important tools for basic and applied biomedical research. Contaminations of cell culture pose a major threat to scientific results. In addition to molds, yeast and bacteria contaminations that are easily detectable by pH shifts, turbidity, and cell destruction, other contaminations such as viruses, mycoplasma and other cell lines are more difficult to detect, and as a result, are a potentially more serious problem. The need for contamination monitoring of any cell lines used for in vitro studies is increasingly emphasized (Lichter et al, 2010, Nature 515:7, Science 346:679) by granting agencies as well as by journals -> no cell-line authentification = no grant/publication. 

The DKFZ has developed a high-throughput multiplex cell contamination test (McCT) currently able to detect >20 contamination markers in a single reaction. 1 (Schmitt et al, 2009)


Non-DKFZ members have access to this service through Multiplexion.


M
ultiplex cell Contamination Testing (McCT) v.2 Service

The McCT v.2 service is designed to simultaneously detect contaminations with: 

  • Mycoplasma (Identification of 14 specific strains: M. arginini, M. fermentans, M. orale,       M. hyorhinis, M. hominis, M. genitalium, M. salivarium, M. synoviae, M. pirum, M. gallisepticum, M. pneumoniae, M. yeatsii, Spiroplasma citri and Acholeplasma laidlawii)
  • Squirrel Monkey Retrovirus (SMRV) and Epstein-Barr virus (EBV)
  • Human cells
  • Monkey cells
  • Mouse cells
  • Rat cells
  • Chinese hamster cells
  • Syrian hamster cells
  • Canine cells
  • Feline cells
  • Rabbit cells
  • Guinea pig cells
  • Pig cells
  • Drosophila S2 cells
  • DNA quality control

    

Test specifications

Detection of contaminations after DNA extraction is done with specific primer sequences in a multiplex PCR targeting viral, bacterial and cellular genome regions, respectively, and subsequent hybridisation using specific oligonucleotide probes. For internal control of DNA quality the presence of the PolA gene is tested. Several positive and negative controls are included to test PCR performance.

Contamination Control Procedure

To be done by the user

  • Cell lysis of cell line(s) by boiling (protocol)
  • Order the test via the submission form
  • Label the Safe Lock tube with “McCT” and the “sample name” and your “cost centre” as provided in the Submission Form
  • Delivery of lysate(s) to the ATV/ICC building INF 242 every Monday, 8-12 a.m. to the doorman

    Please note: The McCT service will be performed on even calendar weeks! But lysates can be delivered weekly every Monday.
    If Monday is a public holiday, lysates delivery is on Tuesdays

 

To be done by the Service Unit

  • Multiplex PCR-based amplification of contaminants
  • Luminex-based detection of PCR products by specific oligonucleotide probes
  • Transfer of results to customer

  

Mycoplasma elimination

We recommend to follow our protocol to eliminate Mycoplasma from your precious cell lines or the protocol of Uphoff and Drexler, 2014

Recommended frequency of testing

According to our own experience, we recommend to control:

  • All batches of stocked cell lines once
  • All new cell lines and new virus cultures
  • Long term-cultured cell lines every month
  • Two weeks after mycoplasma therapy
  • Prior to or concomitant to storage in liquid nitrogen
  • In case of alterations in cell features
  • In case of problems with reproducibility of results

 

Reference: 1 Schmitt, M., and M. Pawlita. 2009. High-throughput detection and multiplex identification of cell contaminations. Nucleic Acids Res 37:e119.

When citing this service, please use the above mentioned reference and indicate the time that passed between the McCT service and the experiments described in your manuscript.

 

Please note: This service is FOR RESEARCH USE ONLY. Do not use in diagnostic procedures

last update:
06/03/2019
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