Why should I be worried about mycoplasma contamination?
Mycoplasma contamination affects up to 80% of continuous cell cultures. If undetected, mycoplasma contamination can have significant effects on the quality and reliability of your cell culture preparations.
- Induces deleterious effects on cell culture quality.
- Alters the phenotypic characteristics of host cells.
- Increases experimental variation.
- Is common in eukaryotic cell cultures.
- Is resistant to antibiotics such as penicillin and streptomycin.
- Has multiple sources (i.e. personnel, reagents, other infected cells).
Mycoplasma are difficult to detect because they:
- Are not visible using standard microscopes.
- Are small enough to pass through 0.45 mm sterilization filters.
- Do not produce changes in culture medium color, pH, or turbidit
What is the best method to determine if my cultured cells are mycoplasma-contaminated?
To provide an accurate and highly sensitive tool for routine screening of mycoplasma contamination in cultured cells, R&D Systems developed the MycoProbe Mycoplasma Detection Assay.
This assay detects Mycoplasma 16S ribosomal RNA (rRNA) using a colorimetric signal amplification system with sensitivity comparable to PCR. The MycoProbe assay is not susceptible to common problems encountered with PCR-based mycoplasma detection kits.
The MycoProbe Mycoplasma Detection Assay:
- Detects the eight mycoplasma species known to cause 95% of eukaryotic cell culture contamination.
- Is highly sensitive (comparable to PCR).
- Is compatible with high-throughput screening.
- Does not generate false positives from amplicon contamination.
- Can be used for cell culture supernatants or cultured cell pellets.
- Can be used with samples from fresh or frozen cells.
- Does not require cells to be cultured in antibiotic-free media.
- Includes a synthetic DNA oligonucleotide positive control.
- Generates results in 4.5 hours.
Alrernative Methods for Mycoplasma Detection
- Takes 2 to 4 weeks
- Cannot detect fastidious mycoplasma
- Requires specialized laboratory conditions
|Fluorescent DNA staining
- Cannot detect mycoplasma that cyto-absorb poorly
|ELISA of cell surface antigens
- Prone to both false positives and false negatives
- Inconsistent results across cell lines
The MycoProbeTM Mycoplasma Detection Kit (Catalog # CUL001B) contains enough reagents to assay one 96-well plate for mycoplasma contamination.
- Cell Lysis Diluent Concentrate - 2 vials (1.7 mL/vial) of a 10-fold concentrated solution
- Hybridization Plate - One 96 well polystyrene microplate
- Streptavidin Plate - One 96 well polystyrene microplate (12 strips of 8 wells) coated with streptavidin
- Sample Diluent - 2 vials (21 mL/vial) of a buffered protein solution with preservatives
- Anti-digoxigenin Conjugate - 21 mL of a polyclonal antibody against digoxigenin conjugated to alkaline phosphatase with preservatives
- Capture Probes - 1.1 mL of a six-fold concentrated stock solution
- Detection Probes - 1.1 mL of a six-fold concentrated stock solution
- Positive Control - 1.1 mL of a solution containing a synthetic DNA oligonucleotide
- Wash Buffer Concentrate - 100 mL of a 10-fold concentrated solution with preservatives
- Substrate - 1 vial of lyophilized NADPH with stabilizers
- Substrate Diluent - 1 vial (7 mL) of a buffered solution with stabilizers
- Amplifier - 1 vial of lyophilized amplifier enzymes with stabilizers
- Amplifier Diluent - 1 vial (7 mL) of a buffered solution containing INT-violet with stabilizers
- Stop Solution - 6 mL of 2 N sulfuric acid
- Float Collar - Microplate float collar for water bath
- Plate Sealers - 12 adhesive strips
The Wash Buffer supplied in this kit contains sodium azide, which may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.
The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
When handling cell culture samples, appropriate precautions should be taken to prevent exposure to mycoplasma and other hazardous biological agents.
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Mycoplasma Detection in Cell Line Supernates and Cell Lysates. The presence of the eight mycoplasma species known to cause 95% of eukaryotic cell culture contamination was tested in supernates and cell lysates of the indicated cell lines using the MycoProbe Mycoplasma Detection Kit (Catalog # CUL001B). The average of the duplicate optical density (OD) readings for each control and sample was determined. The average negative control OD value was subtracted from all average sample OD values. A calculated positive control OD value of >0.10 indicated mycoplasma contamination (black line) in the CTLL-2, BaF3, A431, and K562 (cell lysate 1) samples. Abbreviations: CTLL-2 mouse cytotoxic T cell line, BaF3 mouse pro-B cell line, HepG2 human hepatocellular carcinoma cell line, A431 human epithelial carcinoma cell line, and K562 human chronic myelogenous leukemia cell line.