Caspase 3 devd r110 fluorometric hts assay kit

To load BSA with palmitic acid, ultra-fatty acid free BSA (Sigma-Aldrich) was dissolved in 150 mM NaCl at 37°C to a 0.34 mM concentration and filtered. palmitic acid (Avanti Polar Lipids, Alabaster, AL) was dispersed in 150 mM NaCl with stirring at 70°C for 30 minutes to a 2 mM concentration. Equal volumes of each solution were mixed together, stirred at 37°C for 1 hour and pH was adjusted to 7.4. Aliquots of this stock solution containing 1 mM palmitic acid / 0.17 mM BSA were stored in dark glass vials at -20°C. Identically prepared and stored FA-free 0.17 mM BSA solution was used as the control. To assess toxicity, neuroblastoma cell lines were grown overnight in 96-well plates to ~50% confluency in RPMI media supplemented with 5% FBS. palmitic acid loaded onto BSA and BSA control were added to a final concentration of 0 - 200 uM. Cells were incubated for 24 hours under either normoxic or hypoxic conditions. For hypoxia, cells were incubated in a hypoxia chamber (COY Laboratory Products, Grass Lake, MI) at 1% oxygen. Cell apoptosis was assessed by measuring activity of caspase-3 using Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit (Biotium, Hayward, CA) as recommended by the manufacturer. Readings were obtained via Gen 5 software on a Synergy 2 plate reader (BioTek, Winooski, VT). Each concentration was tested in triplicate in at least three independent experiments.