Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/6/2007
Publication Date: 12/2/2007
Citation: Scherer, A.M., Menge, C., Winter, K., Nystrom, E.A. 2007. Flow cytometric detection of shiga toxin 2 binding to porcine granulocytes in vitro [abstract]. Research Workers in Animal Diseases Conference Proceedings. Paper No. 29P. Interpretive Summary:
Technical Abstract: Granulocytes are hypothesized to be involved in Shiga toxin-producing Escherichia coli (STEC) pathogenesis in humans. Granulocytes bind Shiga toxin (Stx) and may facilitate the transport of Stx to target organs. Pigs are an excellent model for studying the role of granulocytes in STEC disease. Pigs, like humans, are susceptible to Stx-mediated disease and Stx binds to porcine granulocytes in vitro. However, the effects of Stx binding on granulocyte function are unknown. The objectives of this study were 1) to develop a flow cytometric assay for monitoring Stx2 binding to porcine granulocytes in vitro and 2) to optimize the assay for granulocyte functional studies. Porcine granulocytes were isolated from heparinized blood, sequentially incubated with Stx2, anti-Stx2 antiserum, and either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated goat anti-rabbit IgG, and bound Stx2 was detected by flow cytometric analysis. Granulocytes were also incubated with anti-Gb3/CD77 to identify Stx receptors and some experiments included anti-CD172a to confirm that Stx2+ and Gb3/CD77+ cells were granulocytes (>99%). The effects of incubation time, temperature, and buffer on Stx2 binding were evaluated to optimize the assay for granulocyte functional studies. Similar percentages of Stx2+ granulocytes (>98%) and Gb3/CD77 expressing cells (>99%) were detected when the Stx2 incubation step was performed at 4 deg C or 37 deg C. Maximum binding at 37 deg C was observed after 15 minutes. This flow cytometric assay for monitoring Stx2 binding to porcine granulocytes in vitro will facilitate studies to determine the effects of Stx2 on granulocyte function.