Protocol for Detection of IL-8 Activity by Neutrophil Chemotaxis Assay

In response to biological, chemical, or physical factors, human leukocytes produce a series of biologically active (functional) substances, one of which is called interleukins. Many types of interleukins have been identified, interleukin 8 (IL-8) being one of them, and it has recently been discovered that some cells other than leukocytes also can produce interleukins. In this experiment, the migration distance of neutrophils is used to reflect IL-8 activity.

• Prepare 100 mL of 2×DMEM culture solution containing 20 mL of FCS, and 75 mg of NaHCO₃. Place them in a 48°C water bath to pre-warm.
• Prepare 2% agarose and mix it with the above 2×DMEM in equal amounts when holding at about 48°C in a water bath.
• After solidification at room temperature, put it at 4°C for 30-60 min for further solidification, and punch the holes.
• Isolate human peripheral blood neutrophils, and adjust the cell concentration to 2.5×10⁵/mL with DMEM. 10 μL of cell suspension is added to the center well of each group, 10 μL of samples to be examined, and IL-8 or other positive control chemokines are added to the upper well, and 10 μL DMEM is added to the lower well as a negative control.
• The slides are placed in a wet box, incubated at 37°C for 2 h, fixed in methanol for 30 min, stained with Rachel's staining solution, and dried.
• The distance the cells traveled from the edge of the central well toward the sample well (chemotaxis travel distance) and the negative control well (spontaneous travel distance) is measured under the microscope, and the chemotaxis activity is expressed as a chemotaxis index.

Creative Bioarray Relevant Recommendations

• Interleukin-8 (IL-8) is a member of the a (CXC) subfamily of chemokines. It is produced by many cell types, including monocyte/macrophages, T cells, neutrophils, fibroblasts, endothelial cells, keratinocytes, hepatocytes, chondrocytes, and various tumor cell lines, in response to a wide variety of pro-inflammatory stimuli such as exposure to IL-1, TNF, LPS, and viruses. Creative Bioarray provides high-quality Recombinant Human Interleukin-8, Recombinant Human Interleukin 8 (Pichia), Recombinant Human Interleukin 8 (77 aa variant), Recombinant Human Interleukin 8 (72 aa variant), and others.

To confirm that the chemotactic effect in the sample is IL-8 specific, it is also necessary to mix the anti-IL-8 neutralizing antibody with the sample to be tested first, incubate for 1h, then add it to the slide wells and observe the chemotactic reaction. Other relevant antibody-blocking tests can also be used to distinguish the effects of IL-8 and other chemokines.

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