Rat Cytokine Array C1

N° du produit ABIN625675

Détail du produit

Reactivité: Rat

Méthode de détection: Chemiluminescent

Type de méthode: Sandwich ELISA

Application: Antibody Array (AA)

Fonction: C-Series Rat Cytokine Antibody Array 1 Kit. Detects 19 Rat Cytokines. Suitable for all liquid sample types.

Marque: RayBio®

Type d'échantillon: Serum, Plasma, Cell Culture Supernatant, Cell Lysate, Tissue Lysate

Analytical Method: Semi-Quantitative

Specificité: CINC-2, CINC-3, CNTF, Fractalkine (CX3CL1), GM-CSF, IFN-gamma, IL-1 alpha (IL-1 F1), IL-1 beta (IL-1 F2), IL-4, IL-6, IL-10, LIX, Leptin, MCP-1 (CCL2), MIP-3 alpha (CCL20), beta-NGF, TIMP-1, TNF alpha, VEGF-A

Attributs du produit:

• 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

Ingrédients: 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)

Matériel non inclus: 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.

Information d'application

Indications d'application: 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

Commentaires:

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.

Volume d'échantillon: 1 mL

Plaque: Membrane

Protocole:

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

Préparation de l'échantillon:

Use serum-free conditioned media if possible. If serum-containing conditioned media is required, use the serum as a control since many types of sera contain cytokines. For cell lysates and tissue lysates, we recommend using 1X Cell Lysis Buffer to extract proteins from cell or tissue (e.g. using homogenizer). After extraction, spin the sample down and save the supernatant for your experiment. Determine the protein concentration. Dilute 2X Cell Lysis Buffer with H 2 O (we recommend adding proteinase inhibitors to Cell Lysis Buffer before use). We recommend using 1 ml of Conditioned media or 1 ml of original or 10-fold diluted sera or 50-500 µg of protein for cell lysates and tissue lysates. If you experience high background, you may further dilute your sample. 5BU B. Handling Array Membranes. Always use forceps to handle membranes, and grip the membranes by the edges only. Never allow the array membranes to dry during experiments. 6BU C. Incubation. Completely cover the membranes with sample or buffer during incubation, and cover the eight-well tray with a lid to avoid drying. Avoid foaming during incubation steps. Perform all incubation and wash steps under gentle rotation. Several incubation steps such as step 2 (blocking), step 3 (sample incubation), step 7 (biotin-Ab incubation) or step 10 (HRP-streptavidin incubation) may be done at 4 °C for overnight.

Procédure de l'essai:

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.

Calcul des résultats:

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.

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

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: 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.

Stock: -20 °C

Stockage commentaire: 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.

Date de péremption: 6 months

Référence

Shi, Liang, Bao, Xie, Xu, Wang, Wang, Qiao: "Gx-50 Inhibits Neuroinflammation via α7 nAChR Activation of the JAK2/STAT3 and PI3K/AKT Pathways." dans: Journal of Alzheimer's disease : JAD, Vol. 50, Issue 3, pp. 859-71, (2016) (PubMed).

Tsai, Lee, Liu, Chuang, Lu, Dong, Lue, Yang: "Release of endogenous heat shock protein 72 on the survival of sepsis in rats." dans: The Journal of surgical research, Vol. 198, Issue 1, pp. 165-74, (2015) (PubMed).

Yang, Sun, Bhaumik, Li, Baumann, Lin, Zhang, Lin, Dunn, Liu, Shie, Lee, Wills-Karp, Chougnet, Kallapur, Lewkowich, Lindquist, Murali-Krishna, Kuan: "Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic-ischemic brain injury in newborns." dans: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 34, Issue 49, pp. 16467-81, (2014) (PubMed).

Deuse, Hua, Wang, Maegdefessel, Heeren, Scheja, Bolaños, Rakovic, Spin, Stubbendorff, Ikeno, Länger, Zeller, Schulte-Uentrop, Stoehr, Itagaki, Haddad, Eschenhagen, Blankenberg, Kiefmann et al.: "Dichloroacetate prevents restenosis in preclinical animal models of vessel injury. ..." dans: Nature, Vol. 509, Issue 7502, pp. 641-4, (2014) (PubMed).

Busch-Dienstfertig, Labuz, Wolfram, Vogel, Stein: "JAK-STAT1/3-induced expression of signal sequence-encoding proopiomelanocortin mRNA in lymphocytes reduces inflammatory pain in rats." dans: Molecular pain, Vol. 8, pp. 83, (2013) (PubMed).

Holubova, Leba, Sedmikova, Vannucci, Horak: "Characterization of three newly established rat sarcoma cell clones." dans: In vitro cellular & developmental biology. Animal, Vol. 48, Issue 10, pp. 610-8, (2013) (PubMed).

Buijs, Geschwind, Syed, Ganapathy-Kanniappan, Kunjithapatham, Wijlemans, Kook Kwak, Ota, Vali: "Spontaneous tumor regression in a syngeneic rat model of liver cancer: implications for survival studies." dans: Journal of vascular and interventional radiology : JVIR, Vol. 23, Issue 12, pp. 1685-91, (2013) (PubMed).

Kinoshita, Nasu-Tada, Fujishita, Sato, Koizumi: "Secretion of matrix metalloproteinase-9 from astrocytes by inhibition of tonic P2Y14-receptor-mediated signal(s)." dans: Cellular and molecular neurobiology, Vol. 33, Issue 1, pp. 47-58, (2013) (PubMed).

Gruber-Schoffnegger, Drdla-Schutting, Hönigsperger, Wunderbaldinger, Gassner, Sandkühler: "Induction of thermal hyperalgesia and synaptic long-term potentiation in the spinal cord lamina I by TNF-α and IL-1β is mediated by glial cells." dans: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 33, Issue 15, pp. 6540-51, (2013) (PubMed).

Schaal, Garg, Ghosh, Lovera, Lopez, Patel, Louro, Patel, Tuesta, Chan, Pearse: "The therapeutic profile of rolipram, PDE target and mechanism of action as a neuroprotectant following spinal cord injury." dans: PLoS ONE, Vol. 7, Issue 9, pp. e43634, (2012) (PubMed).

Merabova, Kaminski, Krynska, Amini, Khalili, Darbinyan: "JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons." dans: Journal of cellular physiology, Vol. 227, Issue 8, pp. 3119-27, (2012) (PubMed).

Liu, Xu, Chen, Xu, Xiong: "Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity." dans: Cell death & disease, Vol. 3, pp. e254, (2012) (PubMed).

Calvillo, Vanoli, Andreoli, Besana, Omodeo, Gnecchi, Zerbi, Vago, Busca, Schwartz: "Vagal stimulation, through its nicotinic action, limits infarct size and the inflammatory response to myocardial ischemia and reperfusion." dans: Journal of cardiovascular pharmacology, Vol. 58, Issue 5, pp. 500-7, (2012) (PubMed).

Obinata, Matsumoto, Ikado, Sakuma, Kano, Fukuda, Yamaguchi, Mugishima, Takahashi: "Transplantation of mature adipocyte-derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model." dans: International journal of urology : official journal of the Japanese Urological Association, Vol. 18, Issue 12, pp. 827-34, (2012) (PubMed).

Chiu, Chen, Lin, Hsieh, Lin, Cheng: "Study of gastric fluid induced cytokine and chemokine expression in airway smooth muscle cells and airway remodeling." dans: Cytokine, Vol. 56, Issue 3, pp. 726-31, (2012) (PubMed).

Ogawa, de Bold: "Uncoordinated regulation of atrial natriuretic factor and brain natriuretic peptide in lipopolysaccharide-treated rats." dans: Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals, Vol. 17, Issue 2, pp. 140-9, (2012) (PubMed).

Mazo, Cemborain, Gavira, Abizanda, Araña, Casado, Soriano, Hernández, Moreno, Ecay, Albiasu, Belzunce, Orbe, Páramo, Merino, Peñuelas, Verdugo, Pelacho, Prosper: "Adipose stromal vascular fraction improves cardiac function in chronic myocardial infarction through differentiation and paracrine activity." dans: Cell transplantation, Vol. 21, Issue 5, pp. 1023-37, (2012) (PubMed).

Zhang, Yang, Wang, Lin, Lue, Lin: "Adipose tissue-derived stem cells secrete CXCL5 cytokine with neurotrophic effects on cavernous nerve regeneration." dans: The journal of sexual medicine, Vol. 8, Issue 2, pp. 437-46, (2011) (PubMed).

Jung, Kim, Wee, Kim, Choi, Jeong, Lee, Lim, Han: "Bone marrow-derived mesenchymal stromal cells support rat pancreatic islet survival and insulin secretory function in vitro." dans: Cytotherapy, Vol. 13, Issue 1, pp. 19-29, (2011) (PubMed).

Yang, Wu, Zhang, Jin, Wang, Hou, Wei, Hu: "Low-level laser irradiation alters cardiac cytokine expression following acute myocardial infarction: a potential mechanism for laser therapy." dans: Photomedicine and laser surgery, Vol. 29, Issue 6, pp. 391-8, (2011) (PubMed).

Détail du antigène

Sujet: 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.