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Background: Cell Viability/Proliferation Assays

Kit Summary

A complete kit to detect common antibiotic-resistant cell culture contaminants

  • Reduces experimental variation
  • Accurate and highly sensitive
  • Simple multiwell-based colorimetric assay
  • Only takes 4.5 hours
 

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.
See Details

Mycoplasma contamination:

  • 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.   See Details

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

Technique

Advantage

Disadvantage

Microbiological culture Most sensitive
  • Takes 2 to 4 weeks
  • Cannot detect fastidious mycoplasma
  • Requires specialized laboratory conditions
Fluorescent DNA staining Efficient
  • Cannot detect mycoplasma that cyto-absorb poorly
ELISA of cell surface antigens Common technique
  • Low sensitivity
PCR-based detection High sensitivity
  • Prone to both false positives and false negatives
Biochemical activity Common technique
  • Inconsistent results across cell lines

Kit Components

The MycoProbeTM Mycoplasma Detection Kit (Catalog # CUL001B) contains enough reagents to assay one 96-well plate for mycoplasma contamination.   See Details

  • 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

Precautions

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.

 

Data Examples


View Larger Image

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.

Related Research Areas
Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mycoplasma contamination can be evaluated in cell culture supernates or cell pellets using this straightforward procedure:

  • Samples are lysed and hybridized with biotin-labeled capture oligonucleotide probes
  • Digoxigenin-labeled detection probes target the eight most common mycoplasma contaminants
  • Following signal amplification, multiwells are measured using a standard colorimetric plate reader
  • Results are generated in 4.5 hours
 

Reagents Provided

Reagents supplied in the MycoProbe Mycoplasma Detection Kit (Catalog # CUL001B):

  • 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

Other Supplies Required

Reagents

  • Deionized water, RNase-free
  • Cell samples of interest

Materials

  • Pipettes and pipette tips
  • Squirt bottle or manifold dispenser
  • 100 mL and 1,000 mL graduated cylinders for preparation of Wash Buffer
  • Gloves and mask

Equipment

  • Microplate reader capable of measuring absorbance at 490 nm with the correction wavelength set at 650 nm or 690 nm
  • Horizontal orbital microplate shaker (0.12" orbit) capable of maintaining a speed of 500 + 50 rpm
  • 65 + 1 °C water bath
  • Vortex mixer

Procedure Overview

 

 

 

 

 

 
  1. Wash the Hybridization Plate 2 times with Wash Buffer.
  2. Add diluted Probes, Positive Control, Sample Diluent (Negative Control), or sample to the designated wells.
  3. Incubate the plate for 60 minutes in a 65 °C water bath.
  4. Wash the Streptavidin Plate 2 times with Wash Buffer.
  5. Transfer 150 µL from each well of the Hybridization Plate to the Streptavidin Plate.
  6. Incubate for 60 minutes on a horizontal orbital shaker.
  7. Wash the Streptavidin Plate 4 times with Wash Buffer.
  8. Add Anti-Digoxigenin Conjugate to each well.
  9. Incubate for 60 minutes on a shaker.
  10. Wash the Streptavidin Plate 6 times with Wash Buffer.
  11. Add Substrate Solution to each well.
  12. Incubate for 60 minutes on a shaker.
  13. Do not wash.
  14. Add Amplifier Solution to each well.
  15. Incubate for 30 minutes on a shaker.
  16. Do not wash.
  17. Add Stop Solution to each well.
  18. Determine the optical density (OD) of each well within 30 minutes, using a microplate reader set to 490 nm.
  19. Note: If wavelength correction is available, set to 650 nm or 690 nm. If wavelength correction is not available, subtract readings at 650 nm or 690 nm from the readings at 490 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 490 nm without correction may be higher and less accurate.

Calculation of Results

Determine the average of the duplicate optical density (OD) readings for each control and sample. Subtract the average negative control OD value from all average OD values. The calculated positive control OD value should be > 1.5.

OD Values (Calculated)

Result

Interpretation

< 0.05 Negative No mycoplasma detected
0.05 – 0.10 Inconclusive Sample is suspect for mycoplasma. Continue to culture for an additional 2 - 3 days and repeat the test. If sample gives a similar OD, then no mycoplasma are detected.
> 0.10 Positive Mycoplasma detected
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

Showing Results 1 - 8 of 8
Filter your results:

Species
Sample Type
  1. Clonal dominance of CD133+ subset population as risk factor in tumor progression and disease recurrence of human cutaneous melanoma.
    Authors: Sharma, Bhuvnesh, Manglik, V
    Oncogene, 2012;41(5):1570-6.
    Species: Human
  2. Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system.
    Protein Expr. Purif., 2102;84(2):270-9.
    Species: Human
  3. MicroRNA-342 inhibits colorectal cancer cell proliferation and invasion by directly targeting DNA methyltransferase 1.
    Authors: Wang H, Wu J, Meng X
    Carcinogenesis, 2011;32(7):1033-42.
    Species: Human
  4. Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells.
    Authors: Mordant P, Loriot Y, Lahon B, Castier Y, Leseche G, Soria JC, Vozenin MC, Decraene C, Deutsch E
    PLoS ONE, 2011;6(10):e26073.
    Species: Human
  5. Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development.
    Authors: Menghi F, Jacques TS, Barenco M, Schwalbe EC, Clifford SC, Hubank M, Ham J
    Cancer Res., 2011;71(6):2045-55.
    Species: Human
  6. beta2-microglobulin induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells.
    Authors: Josson S, Nomura T, Lin JT, Huang WC, Wu D, Zhau HE, Zayzafoon M, Weizmann MN, Gururajan M, Chung LW
    Cancer Res., 2011;71(7):2600-10.
  7. Interval-specific congenic lines reveal quantitative trait Loci with penetrant lyme arthritis phenotypes on chromosomes 5, 11, and 12.
    Authors: Ma Y, Miller JC, Crandall H, Larsen ET, Dunn DM, Weiss RB, Subramanian M, Weis JH, Zachary JF, Teuscher C, Weis JJ
    Infect. Immun., 2009;77(8):3302-11.
    Species: Mouse
  8. Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.
    Authors: Goto H, Naruto T, Tanoshima R, Kato H, Yokosuka T, Yanagimachi M, Fujii H, Yokota S, Komine H
    Leuk. Res., 2009;33(10):1386-91.
    Species: Human

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