Hank s buffered salt solution hbss buffer

pcDNA 3.1(+) (Invitrogen)

a mammalian expression vector

Restriction enzymes (HindIII, BamHI, KpnI, XhoI)

African green monkey kidney fibroblast-derived COS-7 cells

A 96-well clear bottom microplate (Nunc)

Dulbecco’s modified Eagle’s medium (DMEM)

Fetal bovine serum (FBS; Gibco)

Penicillin/streptomycin (P/S; Gibco)

TransIT-LT1 (Mirus), a lipofection reagent

17β-Estradiol (E₂; native estrogen)

4-Hydroxytamoxifen (OHT; synthetic antiestrogen)

Phosphate-buffered saline (PBS)

A lysis buffer (E291A, Promega)

An assay buffer (E290B, Promega)

Native coelenterazine (nCTZ)

A Bradford reagent for determining total protein amounts

Hank’s buffered salt solution (HBSS) buffer (Gibco)

Grow COS-7 cells in a 6-channel microslide (μ-slide VI^0.4, ibidi) until a 70% fluency.10

Transiently transfect the cells in the channels of the microslide with pErs using a lipofection reagent (TransIT-LT1, Mirus) and keep them in a cell incubator (5% CO₂, 37 °C) for 16 h.

Stimulate the cells in the channels with vehicle (0.1% DMSO) or 10⁻⁶ M OHT for 20 min before image acquisition.

Wash the cells in the channels once with a Hank’s buffered salt solution (HBSS) buffer (Gibco) and then fill the channels simultaneously with 80 μL of an HBSS buffer dissolving nCTZ using a multichannel pipet.

Immediately transfer the microslide into an image analyzer (LAS-4000mini, FujiFilm) and determine the optical images with the equipped software (Image reader ver2.0 and Multi Gauge ver3.1).