Custom-made stably overexpressing hHSD17B13-Myc/DDK HEK293 cells
(LakePharma, Inc.) and estradiol were prepared in serum free Dulbecco’s
modified Eagle’s medium (DMEM) medium containing 10% heat inactivated
FBS, 1× Glutamax, and 1× sodium pyruvate; 24 h prior to
compound testing, 25 μL of a 0.4 × 106 cells/mL
dilution were seeded on 384-well Microplate (culture plate, PerkinElmer,
Cat# 6007680). Compounds were serially diluted in 100% DMSO and 50
nL of the compound dilution were spotted on the preseeded cell plate
by a Labcyte Echo 55× (1% DMSO in the Assay) and incubated for
30 min at 37 °C in a humidified incubator (rH = 95%, CO2 = 5%). After that incubation step, 25 μL of a 60 μM
estradiol dilution were added to each well of the microtiter plate
and incubated for 3 h at 37 °C in the humidified incubator. Finally,
20 μL of supernatant were taken and 2.5 μL of d4-estrone as an internal standard (final conc.
50 nM) were added. To derivatize the analytes, 5 μL of Girard’s
Reagent P (6.5 mM final) dissolved in 90% methanol and 10% formic
acid were added to the samples followed by overnight incubation at
RT before adding 60 μL of dH2O to increase the sample
volume for the RapidFire MS/MS measurements of estrone levels as described
previously. To exclude impact on cell viability as cause for reduced
estrone levels, we also performed a CellTiter-Glo Luminescent Cell
Viability Assay (CellTiter-Glo, Promega, Cat# G9242) with the remaining
cell samples of the initial assay plate, according to manufacturer
protocol. Luminescence was measured using a PHERAstar FSX (BMG Labtech,
Ortenberg, Germany).
(LakePharma, Inc.) and estradiol were prepared in serum free Dulbecco’s
modified Eagle’s medium (DMEM) medium containing 10% heat inactivated
FBS, 1× Glutamax, and 1× sodium pyruvate; 24 h prior to
compound testing, 25 μL of a 0.4 × 106 cells/mL
dilution were seeded on 384-well Microplate (culture plate, PerkinElmer,
Cat# 6007680). Compounds were serially diluted in 100% DMSO and 50
nL of the compound dilution were spotted on the preseeded cell plate
by a Labcyte Echo 55× (1% DMSO in the Assay) and incubated for
30 min at 37 °C in a humidified incubator (rH = 95%, CO2 = 5%). After that incubation step, 25 μL of a 60 μM
estradiol dilution were added to each well of the microtiter plate
and incubated for 3 h at 37 °C in the humidified incubator. Finally,
20 μL of supernatant were taken and 2.5 μL of d4-estrone as an internal standard (final conc.
50 nM) were added. To derivatize the analytes, 5 μL of Girard’s
Reagent P (6.5 mM final) dissolved in 90% methanol and 10% formic
acid were added to the samples followed by overnight incubation at
RT before adding 60 μL of dH2O to increase the sample
volume for the RapidFire MS/MS measurements of estrone levels as described
previously. To exclude impact on cell viability as cause for reduced
estrone levels, we also performed a CellTiter-Glo Luminescent Cell
Viability Assay (CellTiter-Glo, Promega, Cat# G9242) with the remaining
cell samples of the initial assay plate, according to manufacturer
protocol. Luminescence was measured using a PHERAstar FSX (BMG Labtech,
Ortenberg, Germany).
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