Flow cytometric assessment of estrogen receptor β expression in bovine blood neutrophils

The optimisation of a flow cytometric protocol for the determination of the estrogen receptor β (ERβ) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 °C with the primary antibody diluted at 10 μg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 μg/ml. Of the three anti-human or anti-rat ERβ primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERβ protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.