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.