Briefly, B9 cells (5×10³/well of a 96-well plate) are cultured with a series of dilutions of the supernatants in a final volume of 100 mL of RPMI 1640, supplemented with 5×10^-5mol/Lof 2-mercaptoethanol, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in flat-bottom microtiter plates. After 42 h, 0.5 μCi of [³H]thymidine is added. 6 h later, the cells are harvested and the radioactivity incorporated is determined. IL-6 is quantitated from a standard curve set up with known amounts of recombinant human or mouse IL-6. The anti-IL-6 monoclonal antibody completely inhibited the ability of the B9 cells to proliferate in response to recombinant mouse IL-6. In addition, we determined that B9 cells do not proliferate in response to IL-I or TNF, and that antibodies to these two cytokines did not affect the cell proliferation in response to IL-6. Further, it is established that neither Estradiol (17β-estradiol), nor the 0.01% ethanol used as vehicle in these experiments, has any effect on the bioassay.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Adult female Sprague Dawley rats are housed on a 12 hr light/dark cycle with unlimited access to food and water. These animals are left gonadally intact (intact), ovariectomized and treated with estradiol benzoate (OVX+E), or ovariectomized and treated with sesame oil vehicle (OVX+O). The hormone treatment regimen that is used in these experiments has been shown previously to result in differences in CA1 pyramidal cell dendritic spine and synapse density. Six days before electrophysiological analysis, animals are ovariectomized under Metofane anesthesia using aseptic surgical procedure. After surgery, animals are housed individually. On days 3 and 4 after surgery, OVX+E animals are injected (s.c.) with 10 μg of 17β-estradiol benzoate in 100 μL of sesame oil vehicle; OVX+O animalsreceive oil vehicle at each injection time. Forty-eight hours after the second estradiol or vehicle injection, animals are killed by decapitation and hippocampal slices are prepared from their brains. Gonadally intact animals are killed at random stages of the estrous cycle.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Woolley CS, et al. Estradiol increases the sensitivity of hippocampal CA1 pyramidal cells to NMDA receptor-mediated synaptic input: correlation with dendritic spine density. J Neurosci. 1997 Mar 1;17(5):1848-59.

  [2]. Mermelstein PG, et al. Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor. J Neurosci. 1996 Jan 15;16(2):595-604.

  [3]. Girasole G, et al. 17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest. 1992 Mar;89(3):883-91.

  [4]. Woolley CS, et al. Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J Neurosci. 1992 Jul;12(7):2549-54.

  [5]. Woolley CS, et al. Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol. 1993 Oct 8;336(2):293-306.

272.38

C₁₈H₂₄O₂

50-28-2

C[C@@]1([C@H]2O)[C@](CC2)([H])[C@@](CCC3=C4C=CC(O)=C3)([H])[C@]4([H])CC1

Room temperature in continental US; may vary elsewhere