Aposcreen annexin 5 fitc kit

The flow cytometric analysis was performed to analyze the extent of both apoptosis and necrosis of MM cells using an ApoScreen Annexin V-FITC kit (Southern Biotechnology, Birmingham, AL, USA), according to the manufacturer’s protocol. In brief, RPMI 8226 and H929 cells were washed and resuspended in cold binding buffer [10 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), pH 7.4, 140 mM NaCl, 2.5 Mm CaCl2, 0.1% BSA] at cell concentrations between 1 × 106 and 1× 107 cells/ml. After washing, 10 μl labeled annexin V, 380 μl binding buffer and 10 μl PI solution were added to the cell suspension in sequence on ice. Subsequently, the number of stained cells was measured via flow cytometry BD FACScan (Becton Dickinson, San Jose, CA, USA) and analyzed by FlowJo 10.

HeLa cells were seeded at a density of 10⁵ cells per well in 6 well plates and grown overnight, after which media was changed to fresh media containing 10mM 2DG or ¹⁹FDG, 1μg/ml Dox, or a combination of these treatments. Treatment proceeded for 12 hours under either normoxic or hypoxic conditions, after which wells were washed with phosphate buffered saline (PBS) and trypsinized. Neutralized samples were moved into 1.5ml tubes that had been pre-coated at 4°C with 1% BSA/PBS. The ApoScreen^™ Annexin V-FITC Kit (SouthernBiotech) was then used as per manufacturer’s protocol to observe annexin V and propidium iodide staining with the BD FACSCalibur flow cytometer. BD CellQuest Pro was used for analysis.

Cells were harvested after culture and the viability was analyzed by flow cytometry, as previously described²⁶. The percentage of apoptotic cells was calculated from the proportion of neutrophils, as well as lymphocyte positivity for Annexin- V-FITC or propidium iodide (ApoScreen™ Annexin V-FITC kit, SouthernBiotech – Birmingham, AL, USA), in relation to total number of cells²⁶.