Quantification of cell surface antigen expression
The expression of CD11b, ER-α, ER-β, CD14, and TLR4 on the surface of cells was measured by flow cytometry. Cells were treated with fluorescent antibodies (volume per reaction: TLR4 2.5 μL (eBioscience), CD11b 5 μL (BD Bioscience), ER-α 0.5 μL (Abcam), ER-β 5 μL (R&D Systems), CD14 5 μL (eBioscience)). FACS was added and incubated for 10 min at room temperature. The sample was centrifuged at 800 xg for 5 minutes at 4°C. The pellet was resuspended twice with 500 μL DMEM and stored on ice before analysis by flow cytometry and acquired on a BC-CYAN ADP flow cytometer (Beckman Coulter). All measurements were performed under the same instrument settings. A minimum of 5000 events were collected and total cells were gated based on their forward and side scatter characteristics and their positivity of specific fluorochromes (Supplementary Figure). CD14+ monocyte populations were gated and confirmed by cell sorting in flow cytometer gates and expressed as a percentage of total PBMCs. Data were analysed with FlowJo software.