Cytation 5 cell imaging multi mode plate reader

MCF-7 and BT474 cells were seeded at a density of 2 × 10³ cells to a 96-well plate and in the next day, they were treated with 4-OHT (10⁻⁶ M) (Sigma) and SEL (10⁻⁷ M) (Selleckchem) alone and in combination for 24 h. The colorimetric caspase activities of Caspase 2, Caspase 3, Caspase 6, Caspase 8 and Caspase 9 were determined according to the manufacturer recommendations by using ApoTarget Caspase Colorimetric Protease Assay Sampler Kit (Invitrogen, Carlsbad, CA, USA). Changes in colorimetric density were detected by Cytation™ 5 Cell Imaging Multi-Mode Plate Reader (Biotek Instruments, Inc., Winooski, VT, USA) at 400 nm wavelength. Experiments were performed in duplicates and repeated three times.

Cell number was analyzed with Alamar Blue assay at 3 and 7 d. The media was aspirated and scaffolds were washed with D-PBS. A 10 v/v% Alamar Blue solution was prepared by adding Alamar Blue reagent into fully supplemented media and 1.5 mL of 10 v/v% Alamar Blue solution was added to each sample well. Samples were incubated at 37 °C for 2 h, then 300 μL Alamar Blue reagent from each well was transferred in duplicate to a black 96-well plate. Fluorescence measurement was performed using a Cytation5 cell imaging multi-mode plate reader (Biotek, Winooski, VT, USA) at an excitation wavelength of 560 nm and emission wavelength of 590 nm. Cell number was calculated using a standard curve generated from a series of known numbers of cells grown as monolayers. The 231 cell morphologies were observed with fluorescence imaging using a Cytation5 cell imaging multi-mode plate reader.