Scintillation fluid

As described previously [25 (link)], plasma (35–300 μL) was separated from each blood sample after centrifugation and transferred to a glass vial preloaded with scintillation fluid (Fisher Chemical, Waltham, MA, USA). The tracer concentration in the plasma and dose ³H content was then measured by liquid scintillation spectrometry (Beckman Coulter, Brea, CA, USA) to a 2-sigma error of 1% or for a maximum of 300 min.

96-well microtiter filter plates (Millipore, USA) were used in the assay. Each compound was diluted with 1:1 dimethylsulf- oxide assay buffer (50 mM Tris, 10% glycerol, pH 8.0, 0.3 mg/ml ovalbumin, 0.01 M mercaptoethanol) to seven concentrations from 7 × 10^–11 M to 7 × 10^–5 M. 10 μl compound dilutions, 10 μl ³[H]E2 (7 × 10^–8 M) and 50 μl recombinant ER protein (1.0 × 10^–9 M) (PanVera/Invitrogen Corp, Carlsbad, USA) were loaded into each well. The plates were shaken in an orbital shaker for 5 min and incubated overnight (18–24 h) on ice. HAP slurry (Bio- Rad Pacific Ltd., USA) was added and incubated for 15 min at 0°C to capture the protein. The trapped proteins were washed twice with ice cold HAP washing buffer and re-suspended in HAP washing buffer. Portions of the solution were mixed with scintillation fluid (Fisher Scientific, USA) and subjected to measurement by a liquid scintillation counter (Beckman LS6500 Scintillation Counter, USA). The radioactivity of each sample was expressed as disintegration per minute (dpm). The binding of ³[H]E2 to ER in the presence of competitor was determined by subtracting the non-specific binding and expressed as percentage of total binding without competitor. The relative binding affinities (RBA) were calculated by (IC₅₀ 17β-estradiol/IC₅₀ compound) × 100.

Immune cells in supplemented complete RPMI 1640 were plated in 96-well, flat-bottom plates (Falcon, Oxnard, CA, USA) at 2 × 10⁵ cells/well in triplicate without further stimulation. In this study, additional immune challenge was omitted to better mimic in vivo disease conditions. Cultures were maintained for 72 h in a humidified, 7% CO₂ incubator at 37°C. [³H]Thymidine (0.5 μCi/10 μl; Amersham, Arlington Heights, IL, USA) was added the last 24 h of culture. Cells were harvested onto glass fiber filters (Brandel, Gaithersburg, MD, USA) using a cell harvester (Brandel, Gaithersburg, MD, USA). The filters were placed in 5 ml of scintillation fluid (Fisher Scientific, Tustin, CA, USA), and [³H]Thymidine incorporation was determined using a liquid scintillation counter (Beckman, Brea, CA, USA).