Ultra low attachment 96 well culture plate

17β-estradiol (E₂; Calbiochem, Gibbstown, NJ) and progesterone (P; Calbiochem) was dissolved in 100% ethanol at an initial concentration of 1×10⁻³ M, evaporated to dryness in a glass vial and resuspended in complete media consisting of X-VIVO 15 Media (Lonza, Walkersville, MD) supplemented with 10% charcoal stripped human AB serum (Valley Biomedical, Winchester, VA) to a concentration of 1×10⁻⁵ M. Further dilutions were made to achieve final working concentration of 5×10⁻⁸ M E₂ and/or 1×10⁻⁷ M P. Both hormone concentrations are routinely used by our laboratory and are within the physiological range of hormone concentration [24 (link)]. As a control, an equivalent amount of ethanol without dissolved hormone was initially evaporated. Freshly isolated mixed cell suspension were plated at a density of 1×10⁵ cells per well in ultra-low attachment 96-well culture plate (Corning, Corning, NY) in 0.2ml of complete media and treated either with 5×10⁻⁸ M E₂ and/or 1×10⁻⁷ M P at 37°C for 24hr.