Analisi della espressione di citochine in monociti bovini isolati

Cells of monocyte lineage partecipate in immune response by processing and presenting antigens and by producing cytokines, membrane receptors and growth factors that modulate immune response. In order to study cellular interactions in the immune responses of cattles, it is necessary to set up reliable methods to a) obtain highly enriched mononuclear phagocytes populations and b) evaluate the expression ratio of the cyotkines involved in immune response. The present communication describes a method for purify the CD14+ cells from bovine blood, and a procedure to evaluate and quantify by Real Time PCR the espression of cytokines and membrane receptors that are most involved in the modulation of immune response, including IL-1, TNF-alpha, IL-8 receptor, IL-1 beta antagonist, CD18, using GAPDH as housekeeping gene. CD14+ cells have been purified by means of magnetic sorting. The expression of cytokines has been determined after mRNA extraction and reverse trascription using commercial standard procedures and Oligo dT primer. Real Time PCR has been carried out by means of SYBR GREEN technique on iCycler iQ Real-Time PCR Detection System. Relative quantitation of cytokines was calculated as an n-fold difference in transcription of the respective cytokines from control samples (2 - ΔΔCt), using GAPDH as housekeeping gene.