Cell titer glo luminescent viability assay kit

For cell viability assay, cells were seeded at 10,000 cells per well in 96-well plates 1 day before the addition of glimepiride and/or IR for 48 h. Cell viability was determined using CellTiter-Glo® Luminescent Viability Assay kit (Promega). Colony-forming assay was performed following the previous study [35 (link)]. Briefly, the cells were seeded at a density of 600 cells in 35-mm culture dishes. After 24 h, the cells were treated with glimepiride and/or IR. 14 days after seeding, the cells were fixed with 10% methanol and 10% acetic acid, which were then stained with 1% crystal violet. Colonies containing more than 50 cells were identified using densitometry software and scored as survivors.

Luciferase reporter assay was conducted as previously described [18 (link)]. Briefly, MCF-7 cells were seeded at 15,000 cells/well in 96-well plates in phenol red-free medium supplemented with 5% charcoal-stripped FBS (csFBS) (Gibco, A3382101). After 24 h, the cells were transfected with 3× ERE TATA Luc (#11354; Addgene) using Lipofectamine 3000 (L3000001; Invitrogen). The transfected cells were incubated for 5 h and then treated with EAs at 1 μM final concentration. After 24 h of incubation, 50 μl of Promega Bright-Glo luciferase substrate dissolved in lysis buffer (Promega, E2610) was added to the cells. For the cell viability assay, MCF-7 cells were seeded at 15,000 cells/well in 96-well plates in a phenol red-free medium supplemented with 5% csFBS and 10 nM E2. After 24 h, the cells were treated with 1 μM EAs. Cell viability was determined using the CellTiter-Glo Luminescent Viability Assay kit (Promega, G7570) after 48 h of incubation. The luminescence was measured using a CLARIOStar Plus microplate reader.

For the cell viability assay, cells were seeded at 10,000 cells per well in 96-well plates 1 day before the addition of lidoflazine and/or IR for 48 h. Cell viability was determined using a CellTiter-Glo® Luminescent Viability Assay kit (Promega).

Cell Titer-Glo Luminescent Viability Assay kit (Promega Corporation, Madison, WI, USA) was used to monitor cell viability. Control cells or those treated with RAD51 inhibitor and/or etoposide were washed and treated with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. The percentage of cells undergoing apoptosis in each treatment cohort was analyzed by flow cytometry.

Cell Titer-Glo Luminescent Viability Assay kit (Promega Corporation, Madison, WI) was used to assess cell viability.

Cell growth was determined by serial measurement of the number of viable cells after seeding of 2500 cells in opaque-walled 96-well plates, using the Cell-Titer Glo luminescent viability assay kit (Promega, Madison WI). Luminescence intensity was quantified using a BioTek Flx800 plate reader (Winooski, VT). The migration and invasion of MM2BH clones were examined using transwell inserts (8μm pores; Corning, Manassas VA). Uncoated inserts were used in migration, and for the invasion assay, the upper chamber was coated with 30μg of Matrigel (BD Bioscience, San Jose CA). 50,000 cells, suspended in serum-free media, were added to the upper chamber and 10% serum was used as the chemoattractant in lower wells. Migration was continued for 8h and invasion for 64h after cell seeding. Cells were removed from the upper chamber with cotton swabs and cells that migrated on the bottom of the membrane were fixed with chilled methanol for 15min. Subsequently, cells were stained in 1:5000 Hoechst solution (Life Technologies, Eugene OR) for 30min. The membranes were excised and mounted cell-side down on microscope slides using ProLong Gold antifade reagent (Life Technologies, Eugene OR). Ten random fields were imaged at 5x objective magnification on the Zeiss Apotome microscope and the number of cells per field was quantified using the ImageJ software (NIH, Bethesda MD).