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 capture antibodies 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 buffered surfactant with preservative. May turn yellow over time.
C9097-85E: Detection Antibody Cocktail, Mouse Cytokine Array, 1x1vial. Biotinylated antibody cocktail; lyophilized.
C9097-85F: Streptavidin-HRP: To be diluted as directed according to specific batch.
C9097-85G: Chemi Reagent 1, 1x2.5ml. Stabilized hydrogen peroxide with preservative.
C9097-85H: Chemi Reagent 2, 1x2.5ml. Stabilized luminol with preservative.
C9097-85I: 4-Well Multi-dish. Clear 4-well rectangular multi-dish.
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 tips 6. Gloves 7. Deionized or distilled water 8. Rocking platform shaker 9. Microcentrifuge 10. 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 wrap 13. Absorbent lab wipes (KimWipes or equivalent) 14. Paper towels 15. Autoradiography cassette 16. Film developer 17. X-ray film (Kodak, BioMax™ Light-1, Cat. #1788207) 18. Flat-tipped tweezers 19. Flatbed scanner with transparency adapter capable of transmission mode 20. Computer capable of running image analysis software and Microsoft Excel
Other Supplies Required for Cell Lysate Samples
1. Phosphate buffered saline (PBS) 2. Lysis buffer (1% Igepal CA-630, 20mm Tris-HCL (pH 8.0), 137mM NaCl, 10% glycerol, 2mM EDTA, 10ug/ml Aprotinin, 10ug/ml Leupeptin and 10ug/ml Pepstatin
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
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