2.3. Transport Measurements

Uptake of [³H]-estrone sulfate (100 nM) was carried out, following standard protocol previously established in our lab (Wang et al., 2016). Uptake solution (phosphate-buffered saline with 1 mM CaCl₂ and 1 mM MgCl₂, pH 7.4 and 0.3μM [³H]-estrone sulfate) was added to 48-well plates in which cells were plated. The uptake process was terminated by removing the uptake solution and washing the cells with ice-cold PBS twice at indicated time points. 0.2 N NaOH was used for cell lysis and then 0.2 N HCl was used for neutralization. Liquid scintillation counter (Beckman LSC LS6500) was used for detection of [³H]-estrone sulfate. Uptake activity was expressed as a percentage of the uptake measured in control cells. Data were corrected for nonspecific background measured in mock cells (parental cells).