Preliminary studies showed that inclusion of ascorbic acid and selenium in the culture medium enhanced follicular integrity, as evidenced by an increase in the proportion of intact follicles (65% ± 5.9% vs. 35% ± 4.7%; n = 3 experiments; P < 0.05) observable at the end of the 6-day culture period. At the end of the culture period, follicles were embedded in 2% (w/v) agarose, fixed in buffered formalin phosphate solution (10% [v/v], 3 h, RT), stained with Neutral Red (0.1% [w/v], 3 h, RT; to facilitate visualization of follicles during sectioning), and then processed to be embedded in paraffin. Sections (thickness, 4 |xm) of the cultured follicles were also stained with Haematoxylin Phloxine Saffron (HPS) for morphologic examination. plavix 75 mg
Follicular Cell Proliferation
Follicular cell DNA assay. Cultured follicles were washed twice with PBS, fixed and sonicated in trichloroacetic acid (5% [w/v], 20 min, 4°C), and finally washed twice with methanol. The DNA pellet, collected by centrifugation (16 000 X g, 10 min), was dissolved in NaOH (0.25 M) and adjusted to neutral pH with HCl (0.25 M). Aliquots of the dNa pellet and calf thymus DNA (standard) were incubated with Hoechst 33258 dye (0.1 |xg/ml, 5 min in the dark, RT) as described previously. Changes in fluorescence intensity were measured with a Microplate Fluorometer (SPECTRAmax GEMlNlXS, Molecular Devices Corporation, Sunnyvale, CA) at excitation and emission wavelengths of 356 and 457 nm, respectively. The sensitivity and linearity of the assay were 2 and 4-1000 ng/ ml, respectively.