MycoProbe Mycoplasma Detection Kit (CUL001B): R&D Systems
Kit Summary

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

Key Benefits

  • 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.

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. 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.

  • 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.

Preparation and Storage
  • Stability & Storage
    Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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

R&D Systems Protocol for Mouse Treg Cell Differentiation

Wash the Hybridization Plate 2 times with Wash Buffer.

Add diluted Probes, Positive Control, Sample Diluent (Negative Control), or sample to the designated wells.

Incubate the plate for 60 minutes in a 65 °C water bath.

Protocol for Mouse Treg Cell Differentiation Step 1

Wash the Streptavidin Plate 2 times with Wash Buffer.

Transfer 150 µL from each well of the Hybridization Plate to the Streptavidin Plate.

Incubate for 60 minutes on a horizontal orbital shaker.

Protocol for Mouse Treg Cell Differentiation Step 2

Wash the Streptavidin Plate 4 times with Wash Buffer.

Add Anti-Digoxigenin Conjugate to each well.

Incubate for 60 minutes on a shaker.

Protocol for Mouse Treg Cell Differentiation Step 3

Wash the Streptavidin Plate 6 times with Wash Buffer.

Add Substrate Solution to each well.

Incubate for 60 minutes on a shaker.
Do not wash.

Protocol for Mouse Treg Cell Differentiation Step 4

Add Amplifier Solution to each well.

Incubate for 30 minutes on a shaker.
Do not wash.

Protocol for Mouse Treg Cell Differentiation Step 5

Add Stop Solution to each well.

Determine the optical density (OD) of each well within 30 minutes, using a microplate reader set to 490 nm.

Protocol for Mouse Treg Cell Differentiation Step 6

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.

25 Citations: Showing 1 - 10
Filter your results:

Species
Sample Type
  1. Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
    Authors: GS Cho, DI Lee, E Tampakakis, S Murphy, P Andersen, H Uosaki, S Chelko, K Chakir, I Hong, K Seo, HV Chen, X Chen, C Basso, SR Houser, GF Tomaselli, B O'Rourke, DP Judge, DA Kass, C Kwon
    Cell Rep, 2017;18(2):571-582. 2017
  2. BRCA2 hypomorphic missense variants confer moderate risks of breast cancer
    Authors: H Shimelis, RL Mesman, C Von Nicola, A Ehlen, L Guidugli, C Martin, FM Calleja, H Meeks, E Hallberg, J Hinton, J Lilyquist, C Hu, CM Aalfs, K Aittomaki, IL Andrulis, H Anton-Culv, V Arndt, MW Beckmann, JJ Benitez, N Bogdanova, SE Bojesen, MK Bolla, AL Borresen-D, H Brauch, P Brennan, H Brenner, A Broeks, B Brouwers, T Bruning, B Burwinkel, J Chang-Clau, G Chenevix-T, CY Cheng, JY Choi, JM Collée, A Cox, SS Cross, K Czene, H Darabi, J Dennis, T Dork, I Dos Santos, AM Dunning, PA Fasching, JD Figueroa, H Flyger, M Garcia-Clo, GG Giles, G Glendon, P Guenel, CA Haiman, P Hall, U Hamann, M Hartman, FB Hogervorst, A Hollestell, JL Hopper, H Ito, A Jakubowska, D Kang, VM Kosma, V Kristensen, KN Lai, D Lambrechts, L Le Marchan, J Li, A Lindblom, A Lophatanan, J Lubinski, E Machackova, A Mannermaa, S Margolin, F Marme, K Matsuo, H Miao, K Michailido, RL Milne, K Muir, SL Neuhausen, H Nevanlinna, JE Olson, C Olswold, JC Oosterwijk, A Osorio, P Peterlongo, J Peto, PD Pharoah, K Pylkäs, P Radice, MU Rashid, V Rhenius, A Rudolph, S Sangrajran, EJ Sawyer, MK Schmidt, MJ Schoemaker, CM Seynaeve, M Shah, CY Shen, MJ Shrubsole, XO Shu, SL Slager, MC Southey, DO Stram, AJ Swerdlow, SH Teo, I Tomlinson, D Torres, T Truong, CJ van Aspere, LE van der Ko, Q Wang, R Winqvist, AH Wu, JC Yu, W Zheng, Y Zheng, J Leary, LC Walker, L Foretova, F Fostira, K Claes, L Varesco, S Moghadasi, DF Easton, AB Spurdle, P Devilee, H Vrieling, AN Monteiro, DE Goldgar, A Carreira, MP Vreeswijk, FJ Couch
    Cancer Res, 2017;0(0):. 2017
  3. Matrix metalloproteinase processing of PTHrP yields a selective regulator of osteogenesis, PTHrP1-17
    Authors: JS Frieling, G Shay, V Izumi, ST Aherne, RG Saul, M Budzevich, J Koomen, CC Lynch
    Oncogene, 2017;0(0):. 2017
  4. CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer
    Authors: D Wang, H Sun, J Wei, B Cen, RN DuBois
    Cancer Res., 2017;0(0):. 2017
  5. Glucose-dependent regulation of pregnane X receptor is modulated by AMP-activated protein kinase
    Authors: PO Oladimeji, W Lin, CT Brewer, T Chen
    Sci Rep, 2017;7(0):46751. 2017
  6. Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
    Nature, 2016;0(0):. 2016
  7. Quantitative FastFUCCI assay defines cell cycle dynamics at single-cell level
    J. Cell. Sci., 2016;0(0):. 2016
  8. SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP
    Proc Natl Acad Sci USA, 2016;113(38):E5685-E5693. 2016
  9. Targeting ?1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice
    Authors: M Rozo, L Li, CM Fan
    Nat. Med, 2016;22(8):889-96. 2016
  10. Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy
    Elife, 2016;5(0):. 2016
  11. New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate
    Oncotarget, 2016;0(0):. 2016
  12. Long non-coding RNA XIST regulates gastric cancer progression by acting as a molecular sponge of miR-101 to modulate EZH2 expression
    J Exp Clin Cancer Res, 2016;35(1):142. 2016
  13. Human keratinocytes have two interconvertible modes of proliferation.
    Authors: Roshan A, Murai K, Fowler J, Simons B, Nikolaidou-Neokosmidou V, Jones P
    Nat Cell Biol, 2016;18(2):145-56. 2016
  14. Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer
    Sci Rep, 2016;6(0):29384. 2016
  15. The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.
    Authors: DesRochers T, Kuo I, Kimmerling E, Ehrlich B, Kaplan D
    PLoS ONE, 2015;10(3):e0120097. 2015
  16. Characterization of macrophage--cancer cell crosstalk in estrogen receptor positive and triple-negative breast cancer.
    Authors: Hollmen M, Roudnicky F, Karaman S, Detmar M
    Sci Rep, 2015;5(0):9188. 2015
  17. CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.
    Authors: Koh S, Courtin A, Boyce R, Boyle R, Richards F, Jodrell D
    Cancer Res, 2015;75(17):3583-95. 2015
  18. 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. 2012
  19. Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system.
    Protein Expr. Purif., 2012;84(2):270-9. 2012
  20. 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. 2011
  21. 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. 2011
  22. 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. 2011
  23. 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. 2011
  24. 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. 2009
  25. 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. 2009
Expand to show all 25 Citations
Average Rating: 5 (Based on 2 reviews)

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 MycoProbe Mycoplasma Detection Kit CUL001B
: MycoProbe Mycoplasma Detection Kit [CUL001B]

Other Experimental Details

Other Experimental DetailsOur lab has been using this kit for routine Mycoplasma testing of cultures for over 2 years now. The kit is highly sensitive and specific to Mycoplasma. The data is reproducible and more accurate than other tests, the size of the assay is adjustable (the kit comes as 12 8-well strips). You get reliable results in a few hours. Overall, great product and highly recommended.

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