MycoProbe Mycoplasma Detection Kit

Name: MycoProbe Mycoplasma Detection Kit
Brand: R&D Systems
Rating: 5 (3 reviews)

Mycoplasmas Detected: M. hyorhinis, M. arginini, M. fermentans, M. orale, M. pirum, M. hominis, M. salivarium, Acholeplasma laidlawii

(42 citations)

(3 Reviews)

Datasheet / CoA / SDS

Product Details

Assay Procedure

Citations (42)

FAQs

Reviews

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

CUL001B Data Examples
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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

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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.

Product Datasheets

Product Datasheet

COA

Certificate of Analysis Lookup

Certificate of Analysis Request Form

SDS

Preparation and Storage

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Related Research Areas

Cell Cycle

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

View Graphic Procedure

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.

42 Citations: Showing 1 - 10
Filter your results:

Species	Applications	Sample Type
The ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to induce pancreatic ductal adenocarcinoma regression Authors: Y Wallez, CR Dunlop, TI Johnson, SB Koh, C Fornari, JW Yates, S Bernaldo d, A Lau, FM Richards, DI Jodrell Mol. Cancer Ther., 2018;0(0):. 2018 HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome Authors: S di Martino, CA Amoreo, B Nuvoli, R Galati, S Strano, F Facciolo, G Alessandri, HI Pass, G Ciliberto, G Blandino, R De Maria, M Cioce Oncogene, 2018;0(0):. 2018 O-GlcNAcylation of the tumor suppressor FOXO3 triggers aberrant cancer cell growth Authors: H Shin, HJ Cha, K Na, MJ Lee, JY Cho, CY Kim, EK Kim, CM Kang, H Kim, YK Paik Cancer Res., 2018;0(0):. 2018 Integration of distinct ShcA signaling complexes promotes breast tumor growth and tyrosine kinase inhibitor resistance Authors: JR Ha, R Ahn, HW Smith, V Sabourin, S H?bert, E Cepeda Cañ, YK Im, C Kleinman, WJ Muller, J Ursini-Sie Mol. Cancer Res., 2018;0(0):. 2018 TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue Authors: LA Velazquez-, A Perino, V Lemos, M Zietak, M Nomura, TWH Pols, K Schoonjans Nat Commun, 2018;9(1):245. 2018 Effects of Exposure to Acetaminophen and Ibuprofen on Fetal Germ Cell Development in Both Sexes in Rodent and Human Using Multiple Experimental Systems Authors: P Hurtado-Go, RA Anderson, J Macdonald, S van den Dr, K Kilcoyne, A Jørgensen, C McKinnell, S Macpherson, RM Sharpe, RT Mitchell Environ. Health Perspect., 2018;126(4):047006. 2018 Screening, large-scale production and structure-based classification of cystine-dense peptides Authors: CE Correnti, MM Gewe, C Mehlin, AD Bandaranay, WA Johnsen, PB Rupert, MY Brusniak, M Clarke, SE Burke, W De Van Der, K Pilat, SM Turnbaugh, D May, A Watson, MK Chan, CD Bahl, JM Olson, RK Strong Nat. Struct. Mol. Biol., 2018;25(3):270-278. 2018 Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine Authors: SB Koh, Y Wallez, CR Dunlop, S Bernaldo d, TE Bapiro, FM Richards, DI Jodrell Cancer Res., 2018;0(0):. 2018 Smooth muscle glucose metabolism promotes monocyte recruitment and atherosclerosis in a mouse model of metabolic syndrome Authors: VZ Wall, S Barnhart, JE Kanter, F Kramer, M Shimizu-Al, N Adhikari, TN Wight, JL Hall, KE Bornfeldt JCI Insight, 2018;3(11):. 2018 Human Melanoma-Derived Extracellular Vesicles Regulate Dendritic Cell Maturation Authors: RLG Maus, JW Jakub, WK Nevala, TA Christense, K Noble-Orcu, Z Sachs, TJ Hieken, SN Markovic Front Immunol, 2017;8(0):358. 2017 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 HIF2? targeted RNAi therapeutic inhibits clear cell renal cell carcinoma Authors: SC Wong, W Cheng, H Hamilton, AL Nicholas, DH Wakefield, A Almeida, AV Blokhin, J Carlson, ZC Neal, V Subbotin, G Zhang, J Hegge, S Bertin, VS Trubetskoy, DB Rozema, DL Lewis, SB Kanner Mol. Cancer Ther., 2017;0(0):. 2017 CDC27 Induces Metastasis and Invasion in Colorectal Cancer via the Promotion of Epithelial-To-Mesenchymal Transition Authors: L Qiu, X Tan, J Lin, RY Liu, S Chen, R Geng, J Wu, W Huang J Cancer, 2017;8(13):2626-2635. 2017 Lymphatic endothelial progenitors originate from plastic myeloid cells activated by toll-like receptor-4 Authors: LD Volk-Drape, KL Hall, AC Wilber, S Ran PLoS ONE, 2017;12(6):e0179257. 2017 Transcription factor ZNF148 is a negative regulator of human muscle differentiation Authors: J Bakke, WC Wright, AE Zamora, SS Ong, YM Wang, JD Hoyer, CT Brewer, PG Thomas, T Chen Sci Rep, 2017;7(1):8138. 2017 STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4 Authors: H Wang, X Huo, XR Yang, J He, L Cheng, N Wang, X Deng, H Jin, N Wang, C Wang, F Zhao, J Fang, M Yao, J Fan, W Qin Mol. Cancer, 2017;16(1):136. 2017 Lack of constitutively active DNA repair sensitizes glioblastomas to Akt inhibition and induces synthetic lethality with radiation treatment in a p53-dependentmanner Authors: K Palanicham, D Patel, JR Jacob, KT Litzenberg, N Gordon, K Acus, SE Noda, A Chakravart Mol. Cancer Ther., 2017;0(0):. 2017 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 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 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 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 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 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 The orphan nuclear receptor COUP-TFII coordinates hypoxia-independent proangiogenic responses in hepatic stellate cells Authors: Andrea Galli J. Hepatol., 2016;0(0):. 2016 New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate Oncotarget, 2016;0(0):. 2016 SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP Proc Natl Acad Sci USA, 2016;113(38):E5685-E5693. 2016 Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition Nature, 2016;0(0):. 2016 Quantitative FastFUCCI assay defines cell cycle dynamics at single-cell level J. Cell. Sci., 2016;0(0):. 2016 Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy Elife, 2016;5(0):. 2016 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 Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer Sci Rep, 2016;6(0):29384. 2016 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 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 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 Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system. Protein Expr. Purif., 2012;84(2):270-9. 2012 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 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 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 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 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 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 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 Citations

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Average Rating: 5 (Based on 3 reviews)

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: MycoProbe Mycoplasma Detection Kit [CUL001B].

Other Experimental Details

Our 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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MycoProbe Mycoplasma Detection Kit