Maus Cytokine Array C1000

Produktnummer ABIN625629

Produktdetails

Reaktivität: Maus

Nachweismethode: Chemiluminescent

Methodentyp: Sandwich ELISA

Applikation: Antibody Array (AA)

Verwendungszweck: C-Series Mouse Cytokine Antibody Array 1000. A combination of Mouse Cytokine Antibody Array C3 & C4. Detects 96 Mouse Cytokines. Suitable for all liquid sample types.

Marke: RayBio®

Proben: Serum, Plasma, Cell Culture Supernatant, Cell Lysate, Tissue Lysate

Analytische Methode: Semi-Quantitative

Spezifität: Axl, bFGF, BLC (CXCL13), CD30 Ligand (TNFSF8), CD30 (TNFRSF8), CD40 (TNFRSF5), CRG-2, CTACK (CCL27), CXCL16, CD26 (DPPIV), Dtk, Eotaxin-1 (CCL11), Eotaxin-2 (MPIF-2/CCL24), E-Selectin, Fas Ligand (TNFSF6), Fc gamma RIIB (CD32b), Flt-3 Ligand, Fractalkine (CX3CL1), GCSF, GITR (TNFRSF18), GM-CSF, HGFR, ICAM-1 (CD54), IFN-gamma, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IGF-1, IGF-2, IL-1 beta (IL-1 F2), IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-15, IL-17A, IL-17 RB, IL-1 alpha (IL-1 F1), IL-2, IL-3, IL-3 R beta, IL-4, IL-5, IL-6, IL-7, IL-9, I-TAC (CXCL11), KC (CXCL1), Leptin, Leptin R, LIX, L-Selectin (CD62L), Lungkine (CXCL15), Lymphotactin (XCL1), MCP-1 (CCL2), MCP-5, M-CSF, MDC (CCL22), MIG (CXCL9), MIP-1 alpha (CCL3), MIP-1 gamma, MIP-2, MIP-3 beta (CCL19), MIP-3 alpha (CCL20), MMP-2, MMP-3, Osteopontin (SPP1), Osteoprotegerin (TNFRSF11B), Platelet Factor 4 (CXCL4), Pro-MMP-9, P-Selectin, RANTES (CCL5), Resistin, SCF, SDF-1 alpha (CXCL12 alpha), Sonic Hedgehog N-Terminal (Shh-N), TNF RI (TNFRSF1A), TNF RII (TNFRSF1B), TARC (CCL17), I-309 (TCA-3/CCL1), TECK (CCL25), TCK-1 (CXCL7), TIMP-1, TIMP-2, TNF alpha, Thrombopoietin (TPO), TRANCE (TNFSF11), TROY (TNFRSF19), TSLP, VCAM-1 (CD106), VEGF-A, VEGFR1, VEGFR2, VEGFR3, VEGF-D

Produktmerkmale:

• 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

Bestandteile: 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)

Benötigtes Material: 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.

Anwendungsinformationen

Applikationshinweise: 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

Kommentare:

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.

Probenmenge: 1 mL

Plattentyp: Membrane

Protokoll:

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

Aufbereitung der Proben:

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.

Testdurchführung:

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.

Ergebnisberechnung:

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.

Testpräzision: Inter-array Coefficient of Variation (CV) of spot signal intensities as low as 5% when run under optimal conditions.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: 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.

Lagerung: -20 °C

Informationen zur Lagerung: 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.

Haltbarkeit: 6 months

Referenzen

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).

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).

Novitskiy, Forrester, Pickup, Gorska, Chytil, Aakre, Polosukhina, Owens, Yusupova, Zhao, Ye, Shyr, Moses: "Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis." in: Breast cancer research : BCR, Vol. 16, Issue 5, pp. 425, (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).

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).

Braig, Pällmann, Preukschas, Steinemann, Hofmann, Gompf, Streichert, Braunschweig, Copland, Rudolph, Bokemeyer, Koschmieder, Schuppert, Balabanov, Brümmendorf: "A 'telomere-associated secretory phenotype' cooperates with BCR-ABL to drive malignant proliferation of leukemic cells." in: Leukemia, Vol. 28, Issue 10, pp. 2028-39, (2014) (PubMed).

Palmi, Fazio, Savino, Procter, Howell, Cazzaniga, Vieri, Longinotti, Brunati, Andrè, Della Mina, Villa, Greaves, Biondi, DAmico, Ford, Cazzaniga: "Cytoskeletal regulatory gene expression and migratory properties of B-cell progenitors are affected by the ETV6-RUNX1 rearrangement." in: Molecular cancer research : MCR, Vol. 12, Issue 12, pp. 1796-806, (2014) (PubMed).

Stanford, Young, Hicks, Owens, Williams, Vaught, Morrison, Lim, Williams, Brantley-Sieders, Balko, Tonetti, Earp, Cook: "Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution." in: The Journal of clinical investigation, Vol. 124, Issue 11, pp. 4737-52, (2014) (PubMed).

Roth, Kumar, Beer, Richter, Wohlenberg, Reuter, Thiering, Staratschek-Jox, Hofmann, Kreusch, Schultze, Vogl, Roth, Reichelt, Hausser, Magin: "Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18." in: Journal of cell science, Vol. 125, Issue Pt 22, pp. 5269-79, (2013) (PubMed).

Benelli, Stigliani, Minghelli, Carlone, Ferrari: "Impact of CXCL1 overexpression on growth and invasion of prostate cancer cell." in: The Prostate, Vol. 73, Issue 9, pp. 941-51, (2013) (PubMed).

Tavecchio, Lisanti, Lam, Ghosh, Martin, OConnell, Weeraratna, Kossenkov, Showe, Altieri: "Cyclophilin D extramitochondrial signaling controls cell cycle progression and chemokine-directed cell motility." in: The Journal of biological chemistry, Vol. 288, Issue 8, pp. 5553-61, (2013) (PubMed).

Breser, Motrich, Sanchez, Mackern-Oberti, Rivero: "Expression of CXCR3 on specific T cells is essential for homing to the prostate gland in an experimental model of chronic prostatitis/chronic pelvic pain syndrome." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 190, Issue 7, pp. 3121-33, (2013) (PubMed).

Stuelten, Cervoni-Curet, Busch, Sutton, Webster, Kavalukas, Wakefield, Barbul, Niederhuber: "SDF-1α mediates wound-promoted tumor growth in a syngeneic orthotopic mouse model of breast cancer." in: PLoS ONE, Vol. 8, Issue 4, pp. e60919, (2013) (PubMed).

Péqueux, Raymond-Letron, Blacher, Boudou, Adlanmerini, Fouque, Rochaix, Noël, Foidart, Krust, Chambon, Brouchet, Arnal, Lenfant: "Stromal estrogen receptor-? promotes tumor growth by normalizing an increased angiogenesis." in: Cancer research, Vol. 72, Issue 12, pp. 3010-9, (2012) (PubMed).

Capozza, Trimmer, Castello-Cros, Katiyar, Whitaker-Menezes, Follenzi, Crosariol, Llaverias, Sotgia, Pestell, Lisanti: "Genetic ablation of Cav1 differentially affects melanoma tumor growth and metastasis in mice: role of Cav1 in Shh heterotypic signaling and transendothelial migration." in: Cancer research, Vol. 72, Issue 9, pp. 2262-74, (2012) (PubMed).

Chen, Cheung, Reuhl, Liu, Lee, Lu, Yang: "Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice." in: Journal of agricultural and food chemistry, Vol. 59, Issue 21, pp. 11862-71, (2012) (PubMed).

Pan, Jiang, Kantharia, Chen: "A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses." in: Biomedical materials (Bristol, England), Vol. 6, Issue 6, pp. 065002, (2012) (PubMed).

Paulissen, Rocks, Guéders, Bedoret, Crahay, Quesada-Calvo, Hacha, Bekaert, Desmet, Foidart, Bureau, Noel, Cataldo: "ADAM-8, a metalloproteinase, drives acute allergen-induced airway inflammation." in: European journal of immunology, Vol. 41, Issue 2, pp. 380-91, (2011) (PubMed).

Antigendetails

Hintergrund: 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.