Most compounds used in the estrogen receptor library screen were synthesized in-house and have been reported.32 (link)–35 (link), 56 (link), 57 (link) Toremifene, tamoxifen, raloxifene, bazedoxifene, ospemifene, ridaifen-B, nafoxidine, RAD-1901, MPP, acolbifene, and compound 7 were purchased from Sigma-Aldrich or Med Chem Express. All compounds were dissolved in DMSO at a final concentration of 10 mM and were stored at −20 °C until use.
Ospemifene
Manufactured by Merck Group
Ospemifene is a selective estrogen receptor modulator (SERM) used as a laboratory tool for research purposes. It is designed to interact with estrogen receptors in a selective manner. Ospemifene can be used to study the effects of estrogen receptor modulation in various cell-based and in vitro experimental models.
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2 protocols using ospemifene
1
Estrogen Receptor Library Screening
2
Screening Compounds for Gb3 Reduction
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The following drugs were used to perform the assays: Tamoxifen (10 µM, SIGMA 3–48 h), Toremifene (10 µM, SIGMA 48 h) and Ospemifene (10 µM, SIGMA 48 h).
Screening and dose‐response: Cells were plated on 384‐well plates (2 × 104 cells per well). After 24 h, cells were treated with 10 µM compounds or 0.1% dimethyl sulfoxide (DMSO) in complete medium. The Prestwick Library consists of 1,280 FDA‐approved drugs, all off‐patent, dissolved in DMSO. The drugs from the 96‐well source plate were diluted and compacted in 384‐well plates to a concentration of 100 μM in the DMEM medium (working plate). To study the effect of the drugs, 5 μl of the drugs at 100 μM in DMEM medium were added to plates containing 45 μl of medium (10 μM final drug concentration with 0.1% DMSO). As a positive control of Gb3 reduction, we used the glucosylceramide synthase inhibitor PDMP.
For the dose–response confirmation test, compounds were serially diluted from 10 mM stock into complete medium and added to plates starting at 30 to 0.1 µM. The final concentration of DMSO did not exceed 0.3% in the dose–response assays.
Cells were incubated together with drugs 48 h at 37°C and 5% CO2.
Screening and dose‐response: Cells were plated on 384‐well plates (2 × 104 cells per well). After 24 h, cells were treated with 10 µM compounds or 0.1% dimethyl sulfoxide (DMSO) in complete medium. The Prestwick Library consists of 1,280 FDA‐approved drugs, all off‐patent, dissolved in DMSO. The drugs from the 96‐well source plate were diluted and compacted in 384‐well plates to a concentration of 100 μM in the DMEM medium (working plate). To study the effect of the drugs, 5 μl of the drugs at 100 μM in DMEM medium were added to plates containing 45 μl of medium (10 μM final drug concentration with 0.1% DMSO). As a positive control of Gb3 reduction, we used the glucosylceramide synthase inhibitor PDMP.
For the dose–response confirmation test, compounds were serially diluted from 10 mM stock into complete medium and added to plates starting at 30 to 0.1 µM. The final concentration of DMSO did not exceed 0.3% in the dose–response assays.
Cells were incubated together with drugs 48 h at 37°C and 5% CO2.
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