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Detection of Selected Mouse Cytokines in Balb/3T3 Cell Line Lysate. Lysates from Balb/3T3 mouse embryonic fibroblast cells were untreated or treated with 100 ng/mL of recombinant mouse TNF-alpha (R&D Systems, Catalog # 410-MT) for 24 hours (200 µg lysate, 10 minute exposure). |
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Detection of Selected Mouse Cytokines in CMT-93 Cell Line Lysate. Lysates from CMT-93 mouse rectal carcinoma cells were untreated or treated with 100 ng/mL LPS for 24 hours (200 µg lysate, 10 minute exposure). |
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Detection of Selected Mouse Cytokines in Balb/3T3 Cell Line Supernate. Balb/3T3 mouse embryonic fibroblast cells were untreated or treated with 100 ng/mL of recombinant mouse TNF-alpha (R&D Systems, Catalog # 410-MT) for 24 hours (500 µL of cell culture supernate, 5 minute exposure). |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, relative expression levels of mouse cytokines and chemokines in samples can be determined using the following procedure:
- Prepare membrane and incubate with prepared sample
- Incubate the membrane with Detection Antibody Cocktail
- Incubate the membrane array with Streptavidin-HRP
- Develop the membrane array with Chemi Reagents 1 and 2
- Expose the membrane array to autoradiography film
Kit Contents
- Rectangular 4-Well Multi-dish
- 4 Mouse XL Cytokine Array nitrocellulose membranes spotted with 111 different antibodies to mouse cytokines
- Array Buffer 4
- Array Buffer 6
- Chemi Reagent 1
- Chemi Reagent 2
- Detection Antibody Cocktail, Mouse XL Cytokine Array
- Streptavidin-HRP
- Transparency Overlay Template
- Wash Buffer Concentrate (25X)
Other Supplies Required
Reagents
- Aprotinin
- Leupeptin (Catalog #EI002)
- Pepstatin (Catalog #EI003)
- Igepal® CA-630
- Deionized or distilled water
Materials
- Pipettes and pipette tips
- Gloves
- Plastic container with the capacity to hold 50 mL (for washing the arrays)
- Plastic transparent sheet protector (trimmed to 10 cm x 12 cm and open on three sides)
- Plastic wrap
- Absorbent lab wipes (KimWipes® or equivalent)
- Paper towels
- X-ray film (Kodak BioMax™ Light-1) or equivalent
- Flat-tipped tweezers
Equipment
- Rocking platform shaker
- Microcentrifuge
- Autoradiography cassette
- Film developer
- Flatbed scanner with transparency adapter capable of transmission mode
- Computer capable of running image analysis software and Microsoft Excel
Other Supplies Required for Cell Lysate Samples
- Phosphate-Buffered Saline (PBS)
- Lysis buffer (1% Igepal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)
Other Supplies Required for Tissue Lysate Samples
- PBS with protease inhibitors (10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)
- Triton™ X-100
Procedure Overview
R&D Systems Protocol for Multiple Analyte Detection Using the Proteome Profiler™ Mouse XL Cytokine Array Kit, Panel A (Catalog # ARY028)
Add 2 mL of Array Buffer 6 to each well of the supplied 4-Well Multi-dish.
Place each array membrane in a separate well of the 4-Well Multi-dish.
Incubate for one hour on a rocking platform shaker.
Add 0.5 mL Array Buffer 4 to each sample.
Adjust volume of each sample to final volume of 1.5 mL with Array Buffer 6.
Replace the Array Buffer 6 in each well of the 4-Well Multi-dish with prepared samples.
Incubate overnight at 2 °C to 8 °C on a rocking platform.
Wash each array membrane 3 times with 1X Wash Buffer in a separate container.
Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.
Add 30 µL of Detection Antibody Cocktail to 1.5 mL of Array Buffer 4/6 for each array.
Pipette 1.5 mL diluted Detection Antibody Cocktail into each well of the 4-Well Multi-dish.
Return each array membrane to the 4-Well Multi-dish containing diluted Detection Antibody Cocktail.
Incubate for one hour on a rocking platform shaker.
Wash each array membrane 3 times with 1X Wash Buffer in a separate container.
Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.
Add 2 mL of diluted Streptavidin-HRP to each well of the 4-Well Multi-dish.
Place the array membrane in the diluted Streptavidin-HRP solution.
Incubate for 30 minutes on a rocking platform shaker.
Wash each array membrane 3 times with 1X Wash Buffer in a separate container.
Place the array membrane on a plastic sheet protector.
Pipette 1 mL of the prepared Chemi Reagent Mix evenly onto the membrane.
Cover the membrane with the top sheet of the plastic protector.
Incubate for 1 minute.
Blot off excess Chemi Reagent Mix.
Wrap the membrane and sheet protector in plastic wrap.
Place the wrapped array membrane in an autoradiography film cassette and expose to X-ray film.
Citations (34)
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Citations are publications that use Bio-Techne products. Selected citations for Proteome Profiler Mouse XL Cytokine Array include:
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Species: Mouse
Sample Types: Tissue Homogenates
W He et al. (2020), High-salt diet inhibits tumour growth in mice via regulating myeloid-derived suppressor cell differentiation Nat Commun, 11(1):1732.
PMID: 32265505 -
Species: Mouse
Sample Types: Cell Culture Supernates
AA Hartman et al. (2020), YAP Non-cell-autonomously Promotes Pluripotency Induction in Mouse Cells Stem Cell Reports, 14(4):730-743.
PMID: 32243844 -
Species: Mouse
Sample Types: Cell Lysates
AM Decker et al. (2020), Adrenergic Blockade Promotes Maintenance of Dormancy in Prostate Cancer Through Upregulation of GAS6 Transl Oncol, 13(7):100781.
PMID: 32361123 -
Species: Mouse
Sample Types: Serum
D Ren et al. (2020), Sestrin2 modulates cardiac inflammatory response through maintaining redox homeostasis during ischemia and reperfusion Redox Biol, 34(0):101556.
PMID: 32447260 -
Species: Mouse
Sample Types: Serum
Y Cheng et al. (2020), Host cytosolic RNA sensing pathway promotes T Lymphocyte-mediated mycobacterial killing in macrophages PLoS Pathog., 16(5):e1008569.
PMID: 32463840 -
Species: Mouse
Sample Types: Cell Culture Supernates
HL Mercer et al. (2020), Toxoplasma gondii dense granule protein GRA24 drives MyD88-independent p38 MAPK activation, IL-12 production and induction of protective immunity PLoS Pathog., 16(5):e1008572.
PMID: 32413093 -
Species: Mouse
Sample Types: Protein Lysates
P Jiranugrom et al. (2020), NOX4 Deficiency Exacerbates the Impairment of Cystatin C-Dependent Hippocampal Neurogenesis by a Chronic High Fat Diet Genes (Basel), 11(5):.
PMID: 32438638 -
Species: Mouse
Sample Types: Cell Lysates
W Wang et al. (2019), Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion Oncogene, 0(0):.
PMID: 30755731 -
Species: Mouse
Sample Types: Whole Cells
K Haruwaka et al. (2019), Dual microglia effects on blood brain barrier permeability induced by systemic inflammation Nat Commun, 10(1):5816.
PMID: 31862977 -
Species: Mouse
Sample Types: Cell Culture Supernates
A El-Kenawi et al. (2019), Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer Br. J. Cancer, 121(7):556-566.
PMID: 31417189 -
Species: Mouse
Sample Types: Tissue Homogenates
F Hempel et al. (2019), Depletion of Bone Marrow-Derived Fibrocytes Attenuates TAA-Induced Liver Fibrosis in Mice Cells, 8(10):.
PMID: 31591328 -
Species: Mouse
Sample Types: Matrix Bound Nanovesicles
GS Hussey et al. (2019), Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway J Immunol Regen Med, 3(0):26-35.
PMID: 31656879 -
Species: Mouse
Sample Types: Cell Culture Supernates
J Schyns et al. (2019), Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung Nat Commun, 10(1):3964.
PMID: 31481690 -
Species: Mouse
Sample Types: Tissue Homogenates
M Malehmir et al. (2019), Platelet GPIb? is a mediator and potential interventional target for NASH and subsequent liver cancer Nat. Med., 25(4):641-655.
PMID: 30936549 -
Species: Mouse
Sample Types: Plasma
J Liu et al. (2019), Liver-derived fibroblast growth factor 21 mediates effects of glucagon-like peptide-1 in attenuating hepatic glucose output EBioMedicine, 0(0):.
PMID: 30827929 -
Species: Mouse
Sample Types: Serum
P Chang et al. (2018), Mesenchymal stem cells over-expressing cxcl12 enhance the radioresistance of the small intestine Cell Death Dis, 9(2):154.
PMID: 29402989 -
Species: Mouse
Sample Types: Tissue Homogenates
RA Brady et al. (2018), Comparison of the immune response during acute and chronic Staphylococcus aureus infection PLoS ONE, 13(3):e0195342.
PMID: 29596507 -
Species: Mouse
Sample Types: Tissue Homogenates
A Calcinotto et al. (2018), IL-23 secreted by myeloid cells drives castration-resistant prostate cancer Nature, 0(0):.
PMID: 29950727 -
Species: Mouse
Sample Types: Plasma
T Szatmári et al. (2018), Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood Int. J. Radiat. Biol., 0(0):1-31.
PMID: 29533121 -
Species: Mouse
Sample Types: Cell Culture Supernates
KB Gama et al. (2018), Conditioned Medium of Bone Marrow-Derived Mesenchymal Stromal Cells as a Therapeutic Approach to Neuropathic Pain: A Preclinical Evaluation Stem Cells Int, 2018(0):8179013.
PMID: 29535781 -
Species: Mouse
Sample Types: Cell Lysates
T Kobori et al. (2018), Interleukin-18 Amplifies Macrophage Polarization and Morphological Alteration, Leading to Excessive Angiogenesis Front Immunol, 9(0):334.
PMID: 29559970 -
Species: Mouse
Sample Types: Cell Lysates
HR Kim et al. (2018), T cell microvilli constitute immunological synaptosomes that carry messages to antigen-presenting cells Nat Commun, 9(1):3630.
PMID: 30194420 -
Species: Mouse
Sample Types: Cell Culture Supernates
X Sun et al. (2018), Tumor-extrinsic discoidin domain receptor 1 promotes mammary tumor growth by regulatingadipose stromal interleukin 6 production in mice J. Biol. Chem., 0(0):.
PMID: 29298894 -
Species: Mouse
Sample Types: Peritoneal Fluid
MJ Strowitzki et al. (2017), Pharmacological HIF-inhibition attenuates postoperative adhesion formation Sci Rep, 7(1):13151.
PMID: 29030625 -
Species: Mouse
Sample Types: Tissue Homogenates
WN Jin et al. (2017), Depletion of microglia exacerbates postischemic inflammation and brain injury J. Cereb. Blood Flow Metab., 0(0):271678X176941.
PMID: 28273719 -
Species: Mouse
Sample Types: Cell Culture Supernates
C Carmona-Fo et al. (2017), Metabolic origins of spatial organization in the tumor microenvironment Proc. Natl. Acad. Sci. U.S.A, 0(0):.
PMID: 28246332 -
Species: Mouse
Sample Types: Tissue Homogenates
X Lu et al. (2017), Effective combinatorial immunotherapy for castration-resistant prostate cancer Nature, 543(7647):728-732.
PMID: 28321130 -
Species: Mouse
Sample Types: Cell Culture Supernates
D Ribet et al. (2017), Promyelocytic Leukemia Protein (PML) Controls Listeria monocytogenes Infection MBio, 8(1):.
PMID: 28074026 -
Species: Mouse
Sample Types: Cell Lysates
K Murakami et al. (2017), A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro PLoS ONE, 12(7):e0181126.
PMID: 28708884 -
Species: Mouse
Sample Types: Tissue Homogenates
Á Sánchez et al. (2017), Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis Sci Rep, 7(1):5010.
PMID: 28694430 -
Species: Mouse
Sample Types: Cell Culture Supernates
BV Howley et al. (2017), A CREB3-regulated ER-Golgi trafficking signature promotes metastatic progression in breast cancer Oncogene, 0(0):.
PMID: 29249802 -
Species: Mouse
Sample Types: Cell Lysates
(2016), Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity Proc. Natl. Acad. Sci. U.S.A, 0(0):.
PMID: 27994144 -
Species: Mouse
Sample Types: Cell Culture Supernates
Qi Liu (2016), Evaluation of PLGA containing anti-CTLA4 inhibited endometriosis progression by regulating CD4+CD25+Treg cells in peritoneal fluid of mouse endometriosis model Eur J Pharm Sci, 0(0):.
PMID: 27989857 -
Species: Mouse
Sample Types: Peritoneal Lavage Fluid
Nan Chiang (2016), Novel Resolvin D2 Receptor Axis in Infectious Inflammation J. Immunol, 0(0):.
PMID: 27994074
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Customer Reviews (19)
4.8 out of 5
19 customer ratings
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 09/17/2020Excellent antibody array to screen a multitude of cytokines, chemokines, and growth factors in one small strip of membrane. Readout is not a fluorescence so you don't need a special fluorescence reader. If you are doing Western blotting, you can do this easily.
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 02/12/2020Mouse breast tumor tissues
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 01/25/2020 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 01/06/2020Excellent array!!
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 06/06/2019
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 03/13/2019 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 12/05/2018 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 11/11/2018Excellent specificity and detection for large number of cytokines. Works perfectly with little optimization.
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 11/05/2018Pancreas lysate. 750ug protein/ membrane
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 07/18/2018 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 04/01/2018 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 03/22/2018
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 12/07/2017200 uL pooled serum from wild-type C57Bl/6 mice.5 min exposure time
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 08/17/2017 -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 07/17/2017
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 05/23/2017
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 11/28/2016150 uL of mouse serum
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Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 09/30/2016This is a great kit. It provides excellent reliability and a large number of cytokines, chemokines to screening in a single assay. I strongly recommend it. -
Proteome Profiler Mouse XL Cytokine Array
Verified Customer | Posted 08/26/2016The sample was cell culture supernatant collected from MEF.
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FAQs
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Will the Array identification number stamped on the Array membrane interfere with detection if it is not cut-off before the membrane is blocked?
The dye used for printing the Array identification number on the membranes will fluoresce and interfere with the LI-COR detection. It is critical that the number is cut off before beginning the experiment.
