Phone:
+1 877 302 8632
Fax:
+1 888 205 9894 (Toll-free)
E-Mail:
orders@antibodies-online.com

Mouse Cytokine Array C3

Reactivity: Mouse AA Chemiluminescent Semi-Quantitative Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Lysate Membrane
Catalog No. ABIN625641
  • Reactivity
    Mouse
    Detection Method
    Chemiluminescent
    Method Type
    Sandwich ELISA
    Application
    Antibody Array (AA)
    Purpose
    C-Series Mouse Cytokine Antibody Array 3 Kit. Detects 62 Mouse Cytokines. Suitable for all liquid sample types.
    Brand
    RayBio®
    Sample Type
    Serum, Plasma, Cell Culture Supernatant, Cell Lysate, Tissue Lysate
    Analytical Method
    Semi-Quantitative
    Specificity
    Axl, BLC (CXCL13), CD30 Ligand (TNFSF8), CD30 (TNFRSF8), CD40 (TNFRSF5), CRG-2, CTACK (CCL27), CXCL16, Eotaxin-1 (CCL11), Eotaxin-2 (MPIF-2/CCL24), Fas Ligand (TNFSF6), Fractalkine (CX3CL1), GCSF, GM-CSF, IFN-gamma, IGFBP-3, IGFBP-5, IGFBP-6, IL-1 beta (IL-1 F2), IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17A, IL-1 alpha (IL-1 F1), IL-2, IL-3, IL-3 R beta, IL-4, IL-5, IL-6, IL-9, KC (CXCL1), Leptin, Leptin R, LIX, L-Selectin (CD62L), Lymphotactin (XCL1), MCP-1 (CCL2), MCP-5, M-CSF, MIG (CXCL9), MIP-1 alpha (CCL3), MIP-1 gamma, MIP-2, MIP-3 beta (CCL19), MIP-3 alpha (CCL20), Platelet Factor 4 (CXCL4), P-Selectin, RANTES (CCL5), SCF, SDF-1 alpha (CXCL12 alpha), TNF RI (TNFRSF1A), TNF RII (TNFRSF1B), TARC (CCL17), I-309 (TCA-3/CCL1), TECK (CCL25), TIMP-1, TNF alpha, Thrombopoietin (TPO), VCAM-1 (CD106), VEGF-A
    Characteristics
    • Easy to use
    • No specialized equipment needed
    • Compatible with nearly any liquid sample
    • Proven technology (many publications)
    • Highly sensitive (pg/mL)
    • Sandwich ELISA specificity
    • Higher density than ELISA, Western blot or bead-based multiplex
    Components
    Antibody Array Membranes
    Biotinylated Detection Antibody Cocktail
    Blocking Buffer
    Wash Buffers 1 and 2
    Cell & Tissue Lysis Buffer
    Detection Buffers C and D
    Plastic Incubation Tray
    Protease Inhibitor Cocktail (in select kits)
    Material not included
    Pipettors, pipet tips and other common lab consumables
    Orbital shaker or oscillating rocker
    Tissue Paper, blotting paper or chromatography paper
    Adhesive tape or Saran Wrap
    Distilled or de-ionized water
    A chemiluminescent blot documentation system (such as UVP's ChemiDoc-It® or EpiChem II Benchtop Darkroom), X-ray Film and a suitable film processor, or another chemiluminescent detection system.
  • Application Notes
    Perform ALL incubation and wash steps under gentle rotation or rocking motion (~0.5 to 1 cycle/sec) using an orbital shaker or oscillating rocker to ensure complete and even reagent/sample coverage. Rocking/rotating too vigorously may cause foaming or bubbles to appear on the membrane surface which, should be avoided. All washes and incubations should be performed in the Incubation Tray (ITEM 10) provided in the kit. Cover the Incubation Tray with the lid provided during all incubation steps to avoid evaporation and outside debris contamination. Ensure the membranes are completely covered with sufficient sample or reagent volume during each incubation. Avoid forceful pipetting directly onto the membrane, instead, gently pipette samples and reagents into a corner of each well. Aspirate samples and reagents completely after each step by suctioning off excess liquid with a pipette. Tilting the tray so the liquid moves to a corner and then pipetting is an effective method. Optional overnight incubations may be performed for the following step to increase overall spot signal intensities:
    - Sample Incubation
    - Biotinylated Antibody Cocktail Incubation
    - HRP-Streptavidin Incubation
    Comment

    The C-Series arrays feature chemiluminescent signal detection. The antibodies are spotted on nitrocellulose membrane solid supports and are handled in a very similar manner to Western blots.
    All C-Series arrays work on the sandwich ELISA principle, utilizing a matched pair of antibodies: an immobilized capture antibody and a corresponding biotinylated detection antibody.

    Sample Volume
    1 mL
    Plate
    Membrane
    Protocol
    1. Block membranes
    2. Incubate with Sample
    3. Incubate with Biotinylated Detection Antibody Cocktail
    4. Incubate with HRP-Conjugated Streptavidin
    5. Incubate with Detection Buffers
    6. Image with chemiluminescent imaging system
    7. Perform densitometry and analysis
    Sample Preparation

    Use serum-free conditioned media if possible. If serum-containing conditioned media is required, it is highly recommended that complete medium be used as a control since many types of sera contains cytokines. We recommend the following parameters for your samples: 50 to 100 µl of original or diluted serum, plasma, cell culture media, or other body fluid, or 50-500 µg/ml of protein for cell and tissue lysates. If you experience high background or if the fluorescent signal intensities exceed the detection range, further dilution of your sample is recommended.

    Assay Procedure
    1. Place each membrane into the provided eight-well tray (- means the antibody printed side). 2. Add 2 ml 1X Blocking Buffer and incubate at room temperature for 30 min to block membranes. Note: incubation may be done at 4 °C for overnight. 3. Incubate membranes with 1ml of sample at room temperature for 1 to 2 hours. Dilute sample using 1X Blocking Buffer if necessary. Note: We recommend using 1 ml of Conditioned media or 1 ml of original or 10-fold diluted sera or plasma or 50-500 µg of protein for cell lysates and tissue lysates. Dilute the lysate at least 10 folds with 1 X blocking buffer. Note: The amount of sample used depends on the abundance of cytokines. More of the sample can be used if the signals are too weak. If the signals are too strong, the sample can be diluted further. Note: Incubation may be done at 4 °C for overnight. 4. Decant the samples from each container, and wash 3 times with 2 ml of 1X Wash Buffer I at room temperature with shaking. Please allow 5 min per wash. Dilute 20X Wash Buffer I with H 2 O. 5. Wash 2 times with 2 ml of 1X Wash Buffer II at room temperature with shaking. Allow 5 min per wash. Dilute 20X Wash Buffer II with H 2 O. 6. Prepare working solution for primary antibody. Add 100µl of 1X blocking buffer to the Biotin-Conjugated Anti- Cytokines tube. Mix gently and transfer all mixture to a tube containing 2 ml of 1X blocking buffer. Note: the diluted biotin-conjugated antibodies can be stored at 4 °C for 2-3 days. 7. Add 1 ml of diluted biotin-conjugated antibodies to each membrane. Incubate at room temperature for 1-2 hours. Note: incubation may be done at 4 °C for overnight. 8. Wash as directed in steps 4 and 5. 9. Add 2 ml of 1,000 fold diluted HRP-conjugated streptavidin (e.g. add 2 µl of HRP-conjugated streptavidin to 1998 µl 1X Blocking Buffer) to each membrane. Note: Mix the tube containing 1,000X HRP-Conjugated Streptavidin well before use since precipitation may form during storage. 10. Incubate at room temperature for 2 hours. Note: incubation may be done at 4 °C for overnight. 11. Wash as directed in steps 4 and 5.
      Do not let the membrane dry out during detection. The detection process must be completed within 40 minutes without stopping. 1. Proceed with the detection reaction. Add 250µl of 1X Detection Buffer C and 250µl of 1X Detection Buffer D for one membrane, mix both solutions. Drain off excess wash buffer by holding the membrane vertically with forceps. Place membrane protein side up ( - mark is on the protein side top left corner) on a clean plastic sheet (provided in the kit). Pipette the mixed Detection Buffer onto the membrane and incubate at room temperature for 2 minutes. Ensure that the detection mixture is completely and evenly covering the membrane without any air bubbles. 2. Drain off any excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue. Gently place the membrane, protein side up, on a piece of plastic sheet ( - mark is on the protein side top left corner). Cover with another piece of plastic sheet on the array. Gently smooth out any air bubbles. Avoid using pressure on the membrane. 3. Expose the array to x-ray film (we recommend to use Kodak x-omat AR film) and detect signal using film developer. Or the signal can be detected directly from the membrane using a chemiluminescence imaging system. Expose the membranes for 40 seconds and then re-expose the film according to the intensity of signals. If the signals are too strong (background too high), reduce exposure time (e.g. 5-30 seconds). If the signals are too weak, increase exposure time (e.g. 5-20 min or overnight). Or re-incubate membranes overnight with 1x HRP-conjugated streptavidin, and redo detection in the second day. 4. Save membranes in -20° C to -80° C for future reference.
    Calculation of Results

    Visual comparison of array images may be sufficient to see differences in relative protein expression. However, most researchers will want to perform numerical comparisons of the signal intensities (or more precisely, signal densities), using 2-D densitometry. Gel/Blot documentation systems and other chemiluminescent or phosphorescent detection systems are usually sold as a package with compatible densitometry software. Any densitometry software should be sufficient to obtain spot signal densities from your scanned images. One such software program, ImageJ, is available for free from the NIH website along with an array plug-in.

    Assay Precision
    Inter-array Coefficient of Variation (CV) of spot signal intensities as low as 5% when run under optimal conditions.
    Restrictions
    For Research Use only
  • Handling Advice
    The antibody printed side of each membrane is marked by a dash (-) or number (#) in the upper left corner. Do not allow membranes to dry out during the experiment or they may become fragile and break OR high and/or uneven background may occur. Grasp membranes by the corners or edges only using forceps. DO NOT touch printed antibody spots.
    Storage
    -20 °C
    Storage Comment
    For best results, store the entire kit frozen at -20°C upon arrival. Stored frozen, the kit will be stable for at least 6 months which is the duration of the product warranty period. Once thawed, store array membranes and 1X Blocking Buffer at -20°C and all other reagents undiluted at 4°C for no more than 3 months.
    Expiry Date
    6 months
  • Li, Zhou, Zhang, Zheng, He, Ye, Zhang, Qi, He, Yu, Shao, Qiao, Wang: "Inflammatory Molecule,PSGL-1, Deficiency Activates Macrophages to Promote Colorectal Cancer Growth through NFκB Signaling." in: Molecular cancer research : MCR, Vol. 15, Issue 4, pp. 467-477, (2017) (PubMed).

    Wang, Yu, Sunchu, Shoaf, Dang, Zhao, Caples, Bradley, Beaver, Ho, Löhr, Perez: "Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism." in: Aging cell, Vol. 16, Issue 3, pp. 564-574, (2017) (PubMed).

    Palm, Garcia-Faroldi, Lundberg, Pejler, Kleinau: "Activated mast cells promote differentiation of B cells into effector cells." in: Scientific reports, Vol. 6, pp. 20531, (2017) (PubMed).

    Li, Wu, Zhu, Li, Wu, DAngelo, Wang: "EGCG induces G-CSF expression and neutrophilia in experimental sepsis." in: Immunologic research, Vol. 63, Issue 1-3, pp. 144-52, (2016) (PubMed).

    Karakasheva, Waldron, Eruslanov, Kim, Lee, OBrien, Hicks, Basu, Singhal, Malavasi, Rustgi: "CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer." in: Cancer research, Vol. 75, Issue 19, pp. 4074-85, (2016) (PubMed).

    Jin, Chen, Zhang, Xu, Song, Xu, Oudit, Gao, Zhu, Zhong: "Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling." in: Journal of translational medicine, Vol. 13, pp. 255, (2016) (PubMed).

    Achyut, Shankar, Iskander, Ara, Angara, Zeng, Knight, Scicli, Arbab: "Bone marrow derived myeloid cells orchestrate antiangiogenic resistance in glioblastoma through coordinated molecular networks." in: Cancer letters, Vol. 369, Issue 2, pp. 416-26, (2016) (PubMed).

    Peláez-García, Barderas, Batlle, Viñas-Castells, Bartolomé, Torres, Mendes, Lopez-Lucendo, Mazzolini, Bonilla, García de Herreros, Casal et al.: "A proteomic analysis reveals that Snail regulates the expression of the nuclear orphan receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and interleukin 17 (IL-17) to inhibit adipocyte ..." in: Molecular & cellular proteomics : MCP, Vol. 14, Issue 2, pp. 303-15, (2015) (PubMed).

    Ivshina, Alexandrov, Vertii, Doxsey, Richter: "CPEB regulation of TAK1 synthesis mediates cytokine production and the inflammatory immune response." in: Molecular and cellular biology, Vol. 35, Issue 3, pp. 610-8, (2015) (PubMed).

    Vergori, Lauret, Gaceb, Beauvillain, Andriantsitohaina, Martinez: "PPAR? regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice." in: Stem cells (Dayton, Ohio), Vol. 33, Issue 4, pp. 1292-303, (2015) (PubMed).

    Szabolcsi, Celio: "De novo expression of parvalbumin in ependymal cells in response to brain injury promotes ependymal remodeling and wound repair." in: Glia, Vol. 63, Issue 4, pp. 567-94, (2015) (PubMed).

    Reineke, Lloyd: "The stress granule protein G3BP1 recruits protein kinase R to promote multiple innate immune antiviral responses." in: Journal of virology, Vol. 89, Issue 5, pp. 2575-89, (2015) (PubMed).

    Arshad, Deutsch, Vozenin: "Simultaneous irradiation of fibroblasts and carcinoma cells repress the secretion of soluble factors able to stimulate carcinoma cell migration." in: PLoS ONE, Vol. 10, Issue 1, pp. e0115447, (2015) (PubMed).

    Yang, Gorzelanny, Bauer, Halter, Komljenovic, Bäuerle, Borsig, Roblek, Schneider: "Nuclear heparanase-1 activity suppresses melanoma progression via its DNA-binding affinity." in: Oncogene, Vol. 34, Issue 47, pp. 5832-42, (2015) (PubMed).

    Obenauf, Zou, Ji, Vanharanta, Shu, Shi, Kong, Bosenberg, Wiesner, Rosen, Lo, Massagué: "Therapy-induced tumour secretomes promote resistance and tumour progression." in: Nature, Vol. 520, Issue 7547, pp. 368-72, (2015) (PubMed).

    Grum-Schwensen, Klingelhöfer, Beck, Bonefeld, Hamerlik, Guldberg, Grigorian, Lukanidin, Ambartsumian: "S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance." in: BMC cancer, Vol. 15, pp. 44, (2015) (PubMed).

    Llorens-Martín, Jurado-Arjona, Fuster-Matanzo, Hernández, Rábano, Ávila: "Peripherally triggered and GSK-3β-driven brain inflammation differentially skew adult hippocampal neurogenesis, behavioral pattern separation and microglial activation in response to ibuprofen." in: Translational psychiatry, Vol. 4, pp. e463, (2015) (PubMed).

    Kim, Kim, Wen, Park, Park, Lee, Koh, Lee, Koo, Kim: "Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo." in: The Journal of biological chemistry, Vol. 290, Issue 30, pp. 18708-20, (2015) (PubMed).

    Taka, Mazzio, Goodman, Redmon, Flores-Rozas, Reams, Darling-Reed, Soliman: "Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells." in: Journal of neuroimmunology, Vol. 286, pp. 5-12, (2015) (PubMed).

    Cho, Kim, Jeong, Jeong, Jeong, Yoon, Kim, Ahn: "Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages." in: Experimental & molecular medicine, Vol. 46, pp. e70, (2014) (PubMed).

  • Background
    Cytokines play an important role in innate immunity, apoptosis, angiogenesis, cell growth and differentiation. They are involved in interactions between different cell types, cellular responses to environmental conditions, and maintenance of homeostasis. In addition, cytokines are also involved in most disease processes, including cancer and cardiac diseases.
You are here:
Support