Transwell chemotaxis assay

Chemotaxis assays were performed with 24-well Transwell™ supports (Corning, New York, USA) with 8-μm pore membranes. Briefly, MCF-7 cells were grown with or without 1 × 10⁻⁹ M oestradiol, such that both treatments resulted in equal cell numbers at 72 h. Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using a Ficoll-Paque (GE Healthcare, Parramatta, NSW, Australia) gradient. Ethical approval for blood collection was obtained from the Human Ethics Committee of the University of Otago. Blood donors were healthy and gave informed consent according to the University of Otago Human Ethics guidelines. 1 × 10⁶ PBMCs were added to the top chamber of the transwell in a total volume of 100 µL. In the bottom chamber, MCF-7 cells were cultured in RPMI media with DCC serum with or without oestradiol. RPMI media with DCC serum were used as a negative control. Phytohemagglutinin (PHA)-conditioned media, used as a positive control to induce cell migration, were prepared from PBMCs stimulated with 50 µg/ml gentamicin, 50 µM hydroxyurea (both from Sigma, North Ryde, NSW, Australia), 3 mM lithium chloride (Scharlau, Barcelona, Spain) and 2.5 µg/ml phytohemagglutinin P (Gibco, Carlsbad, CA, USA) in a total volume of 600 µL [30]. After 4-h incubation at 37 °C, transmigrated PBMCs were counted with a hemocytometer, incubated with specific antibodies (Table ​(Table1)1) and analysed by flow cytometry. For the blocking assay, parapoxvirus chemokine binding proteins PVNZ 112.3 and 112.6 [31] were added to the media in the lower chamber of the transwell assay at the indicated concentrations.

Panel for flow cytometry