Incucyte s3 instrument

A549 cells and 293 T-ACE2 cells were seeded in a 96-well plate for 24 h at a cell density of 7000 cells/well and 20,000 cells/well, respectively. Then the medium was replaced with 100 µL of complete medium containing ARND at concentrations ranging from 0.625 to 5 µL/mL and incubation was allowed to proceed for 2 h. The medium containing ARND was then discarded and replaced with the pseudovirus inoculum (in a total volume of 275 µL in each well) for infection to proceed for 2 h. The viral inoculum was then removed, and fresh complete medium (200 µL) was added to the cells and incubation was allowed to proceed for another 72 h.
After pseudovirus infection, the medium in a 96-well plate was removed and replaced with PBS for image acquisition under a 10 × objective using the Incucyte^® S3 instrument (Essen Bioscience, Ann Arbor, MI, USA). The images obtained were analyzed using the Incucyte S3 software to calculate the magnitude of the signals of fluorescence. The signal values were normalized by the uninfected cells and infected cells without ARND treatment.

The migratory ability of MIO-M1 cells treated with the four preparations (FF-S, FD-S, FF-PRP, and FD-PRP) was investigated through a scratch wound assay. For this assay, 10⁴ MIO-M1 cells/well were plated in a 96-well Essen ImageLock plate (Essen Bioscience, Ann Harbor, MI, USA) and cultured in DMEM L-Glutamax (Gibco, Thermofisher Scientific, Waltham, MA, USA) with 5% FBS near 100% confluence. Then, the cell monolayer was scratched using a WoundMaker (Essen BioScience, Ann Harbor, MI, USA) device to simultaneously create wounds in each well, washed in PBS, and treated with 100 µL DMEM L-Glutamax medium containing 5% S-FF, S-FD, PRP-FF, and PRP-FD preparations at 37 °C in an incubator with 5% CO₂.
The culture plate was placed in an IncuCyte S3 instrument (Essen BioScience, Ann Harbor, MI, USA) and kept in a dedicated incubator. Then, each wound image per well was automatically recorded with a 10X objective lens every hour for 48 h using the IncuCyte S3/SX1 optical module phase contrast. Images were processed by using the IncuCyte 2019B software to analyze, over time, two integrated measures characterizing the movement behavior. Specifically, the wound confluence was estimated as the wound region area occupied by cells and expressed as a percentage (%), and the wound width was estimated as the distance between wound edges and expressed in micrometers (μm).

MDA-MB-231, Hs578T and MDA-MB-468 cells were plated at a density of 1,500 cells per well in 96-well plates in replicates of 8. At the time of plating, cells were transfected with 10 ng per well of both a YFP expression vector (pcDNA6.2 N-YFP-DEST) and either the ERβ^WT or ERβ^DBD-Mut expression vector (pcDNA4.0) using FuGENE 6 transfection reagent (Promega). Cells were allowed to adhere overnight and subsequently treated with vehicle control or 1 nM E2. Proliferation of YFP positive cells was monitored over a period of 24 h in an IncuCyte® S3 instrument (Essen Bioscience Inc.). Growth rates were determined by calculating the relative YFP area per confluence area following normalization to time zero (start of treatment).