Balen A H, Er J, Rafferty B, Rose M
Department of Endocrinology, Cobbold Laboratories, Middlesex Hospital, London, United Kingdom.
In Vitro Cell Dev Biol Anim. 1995 Apr;31(4):316-22. doi: 10.1007/BF02634007.
We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 x 10(6) cells.ml-1.well-1 in serum-free medium (Dulbecco's modified Eagle's medium/Ham's F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10(-8) M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.
我们已经描述了一种垂体细胞培养的方案及其特性，该培养已成为一种可靠且可重复的生物测定法，用于检测促卵泡激素（FSH）和促黄体生成素（LH）的分泌。该生物测定法用于测量抑制和刺激促性腺激素分泌的因子的生物活性。所使用的方案包括在雌性Wistar大鼠（体重200至250克）的周期随机阶段将其扑杀，并将其垂体细胞以0.4×10⁶个细胞·毫升⁻¹·孔⁻¹的浓度分散在无血清培养基（杜尔贝科改良的伊格尔培养基/哈姆F12混合物，补充有胰岛素和转铁蛋白）中，置于Falcon 3047 24孔培养板中。预培养24小时后，更换培养基，细胞再培养48小时。去除上清液并检测FSH和LH的基础分泌。然后用10⁻⁸ M促性腺激素释放激素（GnRH）刺激细胞4小时，并检测上清液中GnRH刺激的FSH和LH分泌。所有样品均以一式两份（即四份样品）进行检测，这些样品随机添加到培养板中以尽量减少培养板效应。使用随机数表来实现这种随机化。
