Cytokines and chemokines are extracellular signaling molecules that mediate cell-cell communication. They are released from cells and have critical roles in many biological processes such as cellular growth, differentiation, gene expression, migration, immunity and in inflammation. In most biological processes, multiple cytokines operate in a large network, where the action of one cytokine is regulated by the presence or absence of other cytokines. The Mouse Cytokine Array is a rapid, sensitive, and economical tool to simultaneously detect cytokine differences between samples. The relative expression levels of 40 mouse cytokines can be determined without performing numerous immunoassays.
Principle of the Assay:Carefully selected capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell culture supernates, cell lysates, tissue homogenates, serum, or plasma samples are diluted and mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture is then incubated with the Mouse Cytokine Array membrane. Any cytokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Following a wash to remove unbound material, Streptavidin-HRP and chemiluminescent detection reagents are added sequentially. Light is produced at each spot in proportion to the amount of cytokine bound. Refer to the Appendix for a list and coordinates of analytes and controls.
Kit Components:C9097-85A: Mouse Cytokine Array. 4 nitrocellulose membranes each containing 40 different captureantibodies printed in duplicate.
C9097-85B: Array Buffer 4, 1x21ml. Buffered protein base with preservatives. May contain a precipitate
Mix well before and during use.
C9097-85C: Array Buffer 6, 2x21ml. Buffered protein base with preservatives.
C9097-85D: Wash Buffer Concentrate (25X), 2x21ml. 25-fold concentrated solution of bufferedsurfactant with preservative. May turn yellow over time.
C9097-85J: Transparency Overlay Template. 1 transparency overlay template for coordinate reference.
Storage and Stability:Unused kit is stable for 3 months at 4°C. Once opened, C9097-85A may be stored for up to 3 months at 4°C; return unused membranes to the foil pouch containing the dessicant pack and reseal along entire edge of the zip seal. Store C9097-85J at room temperature.
Other Materials Required But Not Supplied:1. Aprotinin (Sigma Cat. #A6279)2. Leupeptin (Sigma Cat. #L8511)3. Pepstatin (Sigma Cat. #P4265)4. Igepal CA-630 (Sigma Cat. #I3021)5. Pipets and pipet tips6. Gloves7. Deionized or distilled water8. Rocking platform shaker9. Microcentrifuge10. A plastic container with the capacity to hold 50ml (for washing the trays)11. Plastic transparent sheet protector (trimmed to 10cm x 12cm and open on three sides)12. Plastic wrap13. Absorbent lab wipes (KimWipes or equivalent)14. Paper towels15. Autoradiography cassette16. Film developer17. X-ray film (Kodak, BioMax™ Light-1, Cat. #1788207)18. Flat-tipped tweezers19. Flatbed scanner with transparency adapter capable of transmission mode20. Computer capable of running image analysis software and Microsoft Excel
Other Supplies Required for Tissue Lysate Samples:1. PBS with protease inhibitors (10ug/ml Aprotinin, 10ug/ml Leupeptin, and 10ug/ml Pepstatin)2. Triton X-100
