Cell Treatment
Cells were thawed gently on ice. Cells were counted, diluted (1 × 105 cells/50 μL) and then incubated for 1 hour at 37°C ± ethanol vehicle, E2 (10 nM) (Sigma-Aldrich), ± the selective ER antagonist ICI 182,780 (10 nM) (Sigma-Aldrich), or both in combination, ± LPS (1 μg/mL) to mimic an inflammatory environment in vitro . 10 nM E2 and ethanol vehicle were made up fresh prior to cell treatment. Previous published data from our group have utilised this method of cord blood LPS stimulation20,21 and cryopreserved cells for immune studies5.