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Role and Gonadotrophic Regulation of X-Linked: MATERIALS AND METHODS(5)

METHODS(5)

Alamar Blue assay. Alamar Blue is a nontoxic metabolic dye and has been used to monitor changes in cell numbers during tissue or primary cell culture in vitro. In the present studies, we have used this assay to monitor the relative increase in cell number during follicular growth under the influence of gonadotropin during a 6-day culture period. The assay was performed as described by Ahmed et al.. To confirm the linear relationship between the changes in Alamar Blue reduction and those of cell number under the experimental conditions of the present studies, Alamar Blue (10% [v/v]) was first tested on primary cultures of rat granulosa cells and human ovarian surface epithelial cancer cell cultures (37°C, 6 h) and, subsequently, on rat ovarian follicle cultures (37°C, 3 h) maintained under a humidified atmosphere of 95% (v/v) air and 5% (v/v) CO2. buy nexium canada pharmacy

Absorbance at wavelengths of 570 and 630 nm was measured by a fluorometer (Model MRX; Dynatech Laboratories Inc., Simi Valley, CA), and this ratio was defined as the cell number equivalence. A linear relationship between ovarian cell number and the extent of Alamar Blue reduction was established with the human epithelial ovarian cancer cell line A2780s (r2 = 0.992) and isolated rat granulosa cells (r2 = 0.980). In addition, a direct correlation was also found between Alamar Blue reduction (follicular cell number equivalence) and DNA content (r2 = 0.850) as well as follicular volume within the range of 2.2-25 nl (diameter, — 160-360 |xm; r2 = 0.933) (Fig. 1). As shown in Figure 2A, an increase in follicular volume above this range, however, was not associated with a proportional increase in follicular Alamar Blue reduction, an observation consistent with the fact that the increase in follicular volume during early development is mainly a consequence of increased cell proliferation whereas that during the late stage is caused by antral enlargement. No significant change in Alamar Blue reduction was evident throughout the 6-day culture period in the absence of the gonadotropin (Fig. 2A). Maximum daily growth rate was observed on Day 3 of culture in the presence of FSH (Fig. 2B).