Cyquant reagent

A proliferation assay was performed using CyQuant Reagent (ThermoFisher), following the supplier’s instructions. Briefly, BSCs were seeded in a 96-well plate at a density of 500 cells/well. Cell culture media consisted of either standard proliferation media or proliferation media with added hemoglobin from bovine blood (Sigma, St Louis, MO, USA) or myoglobin from equine skeletal muscle (Sigma) in concentrations of 1, 3, or 5 mg/mL. Both proteins were provided by the supplier in the oxidized met redox form (metmyoglobin or methemoglobin). Four plates were prepared with 6 replicates per group (n = 6) and single plates were recovered after 1, 3, 5, and 7 days of incubation by aspirating the media and storage at −80 °C. 100 µL media was aspirated and replaced by fresh media after 4 days. When all plates were recovered, CyQuant working solution was prepared by diluting the supplied lysis reagent 1:20 in sterile H₂O, followed by the addition of the dye reagent to a dilution of 1:400. Plates were thawed at room temperature, and 200 µL of CyQuant working solution was added to each well. Fluorescence was measured at an excitation of 480 nm and emission of 520 nm with a spectrophotometer (Synergy H1, Biotek, Winooski, VT, USA). Cell number was calculated with a standard curve of cells seeded at a known density.

To visualize RPTEC/TERT1 in the cell spinpods, 3 µL of 1 mg/mL propidium iodide (PI) and 3 µL of CyQUANT reagent (ThermoFisher) were injected through the silicone port and mixed by gentle rocking of the cell spinpod. Cells were visualized by inverted microscopy in situ using a specialized holder to bring the cell spinpod contents into the proper focal plane (Fig. 2c).

Passage 4 hDF were seeded at 800 cells per well in a 96-well plate with treatment groups containing either basal media supplemented with 1% FBS (− control), 0.75 cm²/mL TPAM in basal media with 1% FBS, or complete media supplemented with 10% FBS (+ control) and cultured for 7 days (means shown represent independent experiments with N = 4, results shown are from two independent experiments). Media was changed once every 2 days and time points were days 1, 3, and 7. CyQuant reagent (ThermoFisher) was added to the wells and fluorescence was assayed using a plate reader (SpectraMax M3; Molecular Device) at 480 nm excitation and 520 nm emission.

Cell survival assay using Cyquant reagent (Invitrogen, C7026) was performed as described (19 (link)). Briefly, 1–4 x 10⁵ cells/mL were seeded in 96 well plates overnight, before treatment with either vehicle or drug for 48–72 h. Cells were harvested, washed and frozen at -80°C overnight. After thawing, Cyquant reagent was added and fluorescence intensity was measured in a spectrometer at 485 nm. Relative percent survival was calculated by dividing the reading from drug-treated cells by the reading from vehicle-treated cells. Median-dose effect analysis and ED50 calculation (50% reduction in cell survival) was performed using CompuSyn software (ComboSyn, Inc.), as described previously (21 (link)).

MCF10A (3.0 × 10³ cells/well), MDA-MB-231 (2.0 × 10³ cells/well), and MDA-MB-468 (3.0 × 10³ cells/well) scrambled shRNA- and shERRFI1-transduced cells were seeded into 96-well clear-bottom black plates (Falcon, 353219) in complete media. Twenty-four hours after seeding, cells were starved in DMEM/F12 with 5% CSS-HS or RPMI with 10% CSS-FBS overnight. The cells were then treated with vehicle or CORT (100 nM; 0.0036% final ethanol concentration). Cell proliferation was measured at the 0 and 72 h hormone treatment timepoint by incubating with CyQuant Reagent (Invitrogen, C35011) for 1 h. Bottom-read fluorescence was then measured with excitation wavelength of 480 nM and emission of 535 nM using EnSight Multimode Plate Reader (PerkinElmer). Each treatment had 5 replicates for each timepoint, and all experiments were repeated twice with similar results.