Estradiol elisa kit

Cells were seeded in charcoal stripped serum (ThermoFisher) media and supplemented with 10 nmol/L Androstenedione (Sigma). Supernatant culture media were evaluated for estrogen production using estradiol ELISA Kit (Cayman Chemical) following the manufacturer's protocol at 412 nm absorbance using an Infinite M100 PRO Quadruple monochromator microplate reader (Tecan). Standard curve and estradiol concentrations were determined with Cayman ELISA competitive analysis tool.

A commercial estradiol ELISA kit (Cayman Chemical) was used to determine plasma E2 levels. Briefly, 100 μL of plasma and standard samples were extracted three times using diethyl ether and dissolved in 100 μL of assay buffer. The extracted samples were assayed in duplicate according to the kit instructions.

KGN cells (2 × 10⁵) were seeded onto six-well plates. After siRNA treatment for 48 h or vector treatment for 72 h, we changed fresh culture medium containing certified charcoal stripped fetal bovine serum (Gibco, Grand Island, NY). Then testosterone (T) (10⁻⁷ mol/l) was added into cells and the culture medium was collected 3 h later. The culture medium was centrifuged at a speed of 12000 rpm/min for 5 min before the concentration of E₂ was detected using Roche electrical chemiluminescence immunoassay after 40 times dilution.
The E₂ concentration of rat serum before hCG administration was detected using Estradiol ELISA Kit (Cayman Chemical, Ann Arbor, MI).

Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [49 (link)]. To detect protein–protein interactions with high selectivity and sensitivity, PLAs were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma Aldrich) according to manufacturer’s protocol. Briefly, cells were fixed and incubated with primary antibodies against IFI16 (sc-8023, Santa Cruz Biotechnology), HIF1α (sc-10790), and PRMT2 (ab154154, Abcam). The cells were incubated with PLA probes and the subsequent ligation and rolling circle amplification were performed. The PLA signals were visualized using a Zeiss LSM 710 confocal microscope (Carl Zeiss).
The amounts of IFNα, IFNβ, and IFNγ protein were measured using commercial ELISA kits (MBS2506739, MBS2513798, and MBS2512904, respectively, MyBioSource) according to the manufacturer’s protocol. The amount of estradiol was measured using Estradiol ELISA kit (#582251 and #501890, Cayman) according to the manufacturer’s protocol.

On each experimental day, one 4–6 mL blood sample was collected in a BD vacutainer serum tube (Fisher Scientific) by a registered nurse allowing measurement of serum estradiol and progesterone levels. After centrifugation, serum was collected and stored at -80 degrees Celsius until assayed. Blood samples were assayed by the New York University Core Clinical Lab. 17β-estradiol concentrations were determined using a commercially available enzyme immunoassay (Estradiol ELISA Kit; Cayman Chemical). Progesterone concentrations were determined using a commercially available enzyme immunoassay (Progesterone ELISA Kit; Cayman Chemical). Determination of serum estradiol levels confirmed that subjects (n = 33) tested during menses and mid-follicular phases had reduced levels of circulating estradiol (mean±sd, 151 ± 130 pg/ml during menses and 176 ± 151 pg/ml during mid-follicular) compared with ovulation (263 ± 244 pg/ml) and luteal phases (228 ± 205 pg/ml). Progesterone levels were also lowest during menses (2424 ± 1997 pg/ml) and mid-follicular phases (2533 ± 1990 pg/ml), higher during ovulation (3276 ± 2621 pg/ml) and highest during the luteal phase (6633 ± 5688 pg/ml), also confirming cycle phases. Each assay was run in duplicate and the results were averaged across the two samples.