MycoProbe Mycoplasma Detection Kit
MycoProbe Mycoplasma Detection 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.
Alternative Methods for Mycoplasma Detection
Technique |
Advantage |
Disadvantage |
---|---|---|
Microbiological culture | Most sensitive |
|
Fluorescent DNA staining | Efficient |
|
ELISA of cell surface antigens | Common technique |
|
PCR-based detection | High sensitivity |
|
Biochemical activity | Common technique |
|
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.
Specifications
Product Datasheets
Scientific Data
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.
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 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
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
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.
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.
Wash the Streptavidin Plate 4 times with Wash Buffer.
Add Anti-Digoxigenin Conjugate to each well.
Incubate for 60 minutes on a shaker.
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.
Add Amplifier Solution to each well.
Incubate for 30 minutes on a shaker.
Do not wash.
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.
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.
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 for MycoProbe Mycoplasma Detection Kit
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.
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The ITIM-Containing Receptor: Leukocyte-Associated Immunoglobulin-Like Receptor-1 (LAIR-1) Modulates Immune Response and Confers Poor Prognosis in Invasive Breast Carcinoma
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Maintenance Therapy for ATM-Deficient Pancreatic Cancer by Multiple DNA Damage Response Interferences after Platinum-Based Chemotherapy
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BET, SRC, and BCL2 family inhibitors are synergistic drug combinations with PARP inhibitors in ovarian cancer
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Mechanical stimulation of human dermal fibroblasts regulates pro-inflammatory cytokines: potential insight into soft tissue manual therapies
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Novel oral plasminogen activator inhibitor?1 inhibitor TM5275 attenuates hepatic fibrosis under metabolic syndrome via suppression of activated hepatic stellate cells in rats
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Inhibition of miR-17~92 Cluster Ameliorates High Glucose-Induced Podocyte Damage
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Comprehensive RNA-Seq profiling of the lung transcriptome of Bashbay sheep in response to experimental Mycoplasma ovipneumoniae infection
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PLoS ONE, 2020-07-08;15(7):e0214497. 2020-07-08
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Loss of the transcription factor MAFB limits beta-cell derivation from human PSCs
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Nat Commun, 2020-06-02;11(1):2742. 2020-06-02
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Toxoplasma gondii dense granule protein GRA24 drives MyD88-independent p38 MAPK activation, IL-12 production and induction of protective immunity
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PLoS Pathog., 2020-05-15;16(5):e1008572. 2020-05-15
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CD73 blockade enhances the local and abscopal effects of radiotherapy in a murine rectal cancer model
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BMC Cancer, 2020-05-12;20(1):411. 2020-05-12
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EIF3H orchestrates Hippo pathway-mediated oncogenesis via catalytic control of YAP stability
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Cancer Res., 2020-04-08;0(0):. 2020-04-08
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MAFB promotes cancer stemness and tumorigenesis in osteosarcoma through a Sox9-mediated positive feedback loop
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Expression of Tryptophan 2,3-Dioxygenase in Metastatic Uveal Melanoma
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&alpha-Linolenic acid but not linolenic acid protects against hypertension: critical role of SIRT3 and autophagic flux
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KANSL2 and MBNL3 are regulators of pancreatic ductal adenocarcinoma invasion
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Neutrophil infiltration and whole-cell vaccine elicited by N-dihydrogalactochitosan combined with NIR phototherapy to enhance antitumor immune response and T cell immune memory
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Centrosome amplification in cancer disrupts autophagy and sensitizes to autophagy inhibition
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Influence of Vitamin D on Corneal Epithelial Cell Desmosomes and Hemidesmosomes
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Invest. Ophthalmol. Vis. Sci., 2019-10-01;60(13):4074-4083. 2019-10-01
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Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation
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Genome-wide miRNA profiling and pivotal roles of miRs 125a-5p and 17-92 cluster in human neutrophil maturation and differentiation of acute myeloid leukemia cells
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Chloramphenicol Mitigates Oxidative Stress by Inhibiting Translation of Mitochondrial Complex I in Dopaminergic Neurons of Toxin-Induced Parkinson's Disease Model
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Activation of NIX-mediated mitophagy by an interferon regulatory factor homologue of human herpesvirus
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Aberrant expression of ERG promotes resistance to combined PI3K and AR pathway inhibition through maintenance of AR target genes
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Investigation of Genetic Susceptibility to Blastomycosis Reveals Interleukin-6 as a Potential Susceptibility Locus
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MBio, 2019-06-18;10(3):. 2019-06-18
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Dysregulated Tgfbr2/ERK-Smad4/SOX2 signaling promotes lung squamous cell carcinoma formation
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Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells
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CD38-driven mitochondrial trafficking promotes bioenergetic plasticity in multiple myeloma
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Cancer Res., 2019-01-08;0(0):. 2019-01-08
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Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling
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Elife, 2018-12-27;7(0):. 2018-12-27
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miR-663a inhibits tumor growth and invasion by regulating TGF-?1 in hepatocellular carcinoma
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De novo NAD+ synthesis enhances mitochondrial function and improves health
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The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer
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Targeting PARP1 in XRCC1 deficient sporadic invasive breast cancer or pre-invasive ductal carcinoma in situ induces synthetic lethality and chemoprevention
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Lineage dynamics of murine pancreatic development at single-cell resolution
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Raman micro-spectroscopy for accurate identification of primary human bronchial epithelial cells
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Complex bile duct network formation within liver decellularized extracellular matrix hydrogels
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Sci Rep, 2018-08-15;8(1):12220. 2018-08-15
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The ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to induce pancreatic ductal adenocarcinoma regression
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Mol. Cancer Ther., 2018-06-11;0(0):. 2018-06-11
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Smooth muscle glucose metabolism promotes monocyte recruitment and atherosclerosis in a mouse model of metabolic syndrome
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JCI Insight, 2018-06-07;3(11):. 2018-06-07
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Mechanistic distinctions between CHK1 and WEE1 inhibition guide the scheduling of triple therapy with gemcitabine
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Cancer Res., 2018-05-07;0(0):. 2018-05-07
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Effects of Exposure to Acetaminophen and Ibuprofen on Fetal Germ Cell Development in Both Sexes in Rodent and Human Using Multiple Experimental Systems
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A Novel Strategy to Prevent Advanced Atherosclerosis and Lower Blood Glucose in a Mouse Model of Metabolic Syndrome
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Diabetes, 2018-02-26;0(0):. 2018-02-26
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Screening, large-scale production and structure-based classification of cystine-dense peptides
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Integration of distinct ShcA signaling complexes promotes breast tumor growth and tyrosine kinase inhibitor resistance
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A combination of SAHA and Quinacrine is effective in inducing cancer cell death in upper gastrointestinal cancers
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Clin. Cancer Res., 2018-01-31;0(0):. 2018-01-31
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TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue
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HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome
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O-GlcNAcylation of the tumor suppressor FOXO3 triggers aberrant cancer cell growth
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Cancer Res., 2018-01-04;0(0):. 2018-01-04
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HIF2? targeted RNAi therapeutic inhibits clear cell renal cell carcinoma
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Mol. Cancer Ther., 2017-10-27;0(0):. 2017-10-27
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Lack of constitutively active DNA repair sensitizes glioblastomas to Akt inhibition and induces synthetic lethality with radiation treatment in a p53-dependentmanner
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Mol. Cancer Ther., 2017-08-24;0(0):. 2017-08-24
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CDC27 Induces Metastasis and Invasion in Colorectal Cancer via the Promotion of Epithelial-To-Mesenchymal Transition
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J Cancer, 2017-08-21;8(13):2626-2635. 2017-08-21
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Transcription factor ZNF148 is a negative regulator of human muscle differentiation
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Sci Rep, 2017-08-15;7(1):8138. 2017-08-15
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STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4
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Lymphatic endothelial progenitors originate from plastic myeloid cells activated by toll-like receptor-4
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PLoS ONE, 2017-06-09;12(6):e0179257. 2017-06-09
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CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer
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Cancer Res., 2017-04-28;0(0):. 2017-04-28
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Glucose-dependent regulation of pregnane X receptor is modulated by AMP-activated protein kinase
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Sci Rep, 2017-04-24;7(0):46751. 2017-04-24
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Matrix metalloproteinase processing of PTHrP yields a selective regulator of osteogenesis, PTHrP1-17
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Oncogene, 2017-04-03;0(0):. 2017-04-03
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Human Melanoma-Derived Extracellular Vesicles Regulate Dendritic Cell Maturation
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BRCA2 hypomorphic missense variants confer moderate risks of breast cancer
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Cancer Res, 2017-03-10;0(0):. 2017-03-10
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Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy
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New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate
Oncotarget, 2017-01-03;0(0):. 2017-01-03
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Quantitative FastFUCCI assay defines cell cycle dynamics at single-cell level
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Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy
Elife, 2016-11-18;5(0):. 2016-11-18
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The orphan nuclear receptor COUP-TFII coordinates hypoxia-independent proangiogenic responses in hepatic stellate cells
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Long non-coding RNA XIST regulates gastric cancer progression by acting as a molecular sponge of miR-101 to modulate EZH2 expression
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SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP
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Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition
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Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer
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Targeting ?1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice
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Human keratinocytes have two interconvertible modes of proliferation.
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CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.
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The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.
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Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells.
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Interval-specific congenic lines reveal quantitative trait Loci with penetrant lyme arthritis phenotypes on chromosomes 5, 11, and 12.
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Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.
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FAQs
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What are recommendations for cell culture numbers prior to evaluating supernatant in the kit?
For adherent cells, we recommend culturing cells to confluence prior to the assay. For suspension cells, we recommend culturing to a density of 0.5-1 x 10^6 cells/mL.
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