Introduction

Cytokines are signaling molecules that have critical functions in many biological processes such as cell growth, differentiation, gene expression, migration, immunity, and inflammation. Cytokines are secreted from cells and bind
to cell surface receptors, which initiate the activation of signal transduction pathways and mediate cell-to-cell
communication. Cytokine malfunction leads to many diseases, including arthritis, acute and chronic liver
disease, inflammatory bowel disease, related to heart diseases and cancers.

A group of cytokines commonly implies in a biological or disease process, therefore, the integral analysis of the expression of multiples cytokines reveals the underlying mechanism of the disease state effectively. The mouse cytokine ELISA Plate Array I allows you to control the abundance of 24 mouse cytokines in a high-throughput manner. This essay is a fast and sensitive tool for quantitatively profiling the levels of multiple cytokines between samples simultaneously.

Test principle

The 96-well clear plate is divided into 4 sections, and each section has 3 strips for one sample. In each section, 24 specific cytokine capture antibodies are coated in 24 wells respectively. The sample, such as cell culture supernatants, cell lysates, tissue homogenates, serum or plasma samples are incubated with a cytokine ELISA plate,
and the captured cytokine proteins are subsequently detected with biotinylated detection cocktail antibodies.

The test sample is allowed to react with pairs of two antibodies, resulting in cytokines being sandwiched between the solid phase and enzyme-linked antibodies. After incubation, the wells are washed to remove unbound labeled antibodies. An HRP substrate, TMB, is added to result in the development of a blue color. The color development is then stopped with the addition of Stop the solution by changing the color to yellow. The Cytokine concentrations are directly proportional to the intensity of the color of the test sample. The absorbance is measured spectrophotometrically at 450 nm.

Preparing reagents before starting to experience

• Dilute Assay Wash Buffer 5x in 1x Buffer
  – 40 ml 5x Assay Wash Buffer
  – 160 ml of ddH2O
• Dilute the biotin-labeled antibody mixture 50 times with 1X diluent buffer. (AVOID FREEZING / THAWING THE ANTIBODY MIXTURE)
• Dilute streptavidin-HRP 200 times with 1X. Diluent buffer.

Sample preparation before starting to experience

• For cell culture medium samples, add 100 ul directly to the well or dilute 2 times with 1X diluent buffer.
• For cell lysate samples, use cell lysis buffer (Catalog # EA-0001). Follow the protocol in Cell Lysate Buffer User Manual on our website.
• For serum or plasma samples, we recommend a 1:10 to 1:20 dilution with 1X diluent buffer. When Conditional media containing serum is required, be sure to use the serum as a control