Equine TNF alpha Bioactivity

Kingfisher Biotech's Equine TNFα (cat # RP0137E) was determined to be biological active by an independent laboratory in Spain. The principle of the assay is that Equine TNFα should stimulate an increase in production of IL-6 and iNOS in horse adult mesenchymal cells in culture. The Equine TNFα protein was reconstituted in 1 ml of PBS + 0.1% BSA (bovine serum albumin). The horse adult mesenchymal cells were cultured in 48 well plates, in the presence of TNFα (diluted 1:100) at a final concentration of 25 ng/ml. The cultures were maintained for 12 hours at 37 degrees C. RT-PCR and quantitative PCR (real time PCR) were used to analyze in the supernants of the cultures for IL-6 and the iNOS production. Compared to control cells, the cells stimulated with Equine TNFα had significantly higher levels of IL-6 and iNOS, showing that the Equine TNFα is biologically active.