Annexin 5 dead cell reagent

For the analysis of flow cytometric, MDA-MB-231 cells, at an amount of 5 × 10⁵ cells per well, were plated in six well plates and allowed to stand for 24 and 48 h to obtain a logarithmic growth. The cells were incubated in a complete culture medium containing 10, 25, 50 and 100 µM of the tested compounds for 24 and 48 h. For the apoptotic assay, 1 × 10⁶ of cells in suspension were transferred to the new tube and incubated with 100 µL of Annexin V & Dead Cell reagent (Merck Millipore, Warsaw, Poland) for 20 min at room temperature. The apoptosis was determined with a Muse Cell Analyzer (Warsaw, Poland); emission max.: yellow- red- 576 nm and 680 nm, excitation max.: 532 nm).

The effects of temsirolimus and chemotherapy on the cell cycle and apoptosis were evaluated by seeding tumor cells into 6-well plates at a density of 5 × 10⁴ per well. The cells were treated accordingly and incubated for 24 h followed by a phosphate-buffered saline (PBS) wash. The cell cycle phases were determined using a Muse cell analyzer (Merck Millipore, Darmstadt, Germany) and a Muse Cell Cycle Assay Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Cell apoptosis was analyzed using Annexin V Dead cell reagent (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. An average of at least 10,000 cells was analyzed for each condition. Triplicate independent experiments were conducted.