Cytotoxicity Assay of FD-895 and Pladienolide B

Normal PBMCs were treated with FD-895 (100 nM to 2.0 μM), and pladienolide B (100 nM to 2.0 μM), for 48 h. Cell viability was determined by flow cytometry after staining with conventional live staining with 40 μM 3,3′dihexyloxacarbocyanine iodide (DiOC₆; Life Technologies, Carlsbad, CA, USA) and 15 μM (10 μg/mL) of propidium iodide (PI; Sigma-Aldrich, St Louis, MO, USA). Data were analyzed by using FlowJo software (version 6.4.7; Tree Star). Using this assay, viable cells excluded PI and stained brightly positive for DiOC₆ as it targets metabolically active mitochondria of alive cells [30 (link)–33 (link)].