A cell migration assay was used to measure the effect of hIL-8 on human neutrophil cells. The neutrophils were isolated and loaded into an upper chamber over a permeable membrane at a concentration of 2x106 cells/mL. The hIL-8 was loaded into the bottom chamber at a concentration of 10 ng/mL to induce cell migration. PBS was used as a vehicle control. The chambers were incubated for an hour at 37oC and 5% CO2 prior to being collected and counted on a hemocytometer to measure the percentage of cell migration.
The purity of recombinant hIL-8 was determined by SDS-PAGE of 1.5 µg reduced (+) and non-reduced (-) recombinant hIL-8 and staining overnight with Coomassie Blue.
Recombinant human IL-8 is supplied as lyophilized material that is very stable at -20°C. It is recommended to reconstitute with sterile water at a concentration of 0.1 mg/mL, which can be further diluted in aqueous solutions as needed. Addition of a carrier protein (0.1% HSA or BSA) is recommended for long term storage.
Recombinant human IL-8 was expressed in E. coli and is supplied in a lyophilized form. A greater than 95% purity was determined by SDS-PAGE. Endotoxin levels are less than or equal to 1 EU / 1 μg hIL-8.
The prototypical CXC chemokine, IL-8, is best known for effects on neutrophils, specifically its ability to act as a chemoattractant and activate degranulation and respiratory burst (1,2,3).
IL-8 is produced by a number of cell types, including monocytes, T cells, neutrophils, fibroblasts, endothelial cells, and others (1). In addition to its effects on neutrophils, IL-8 promotes angiogenesis, inhibits endothelial cell apoptosis, and promotes the proliferation of melanoma cells in an autocrine fashion (1). As a result, IL-8 may be a contributing factor to the development of certain cancers (1,2). There are two distinct receptors for IL-8, CXCR1 and CXCR2; both are G protein-coupled receptors (1). Ligand binding induces Ca2+ mobilization and activates the PI3K, PKC, Rho, and Rac pathways (1,3).
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