Cytotox 96 nonradioactive

LDH assay is another determinant for the potential cytotoxicity exerted by PdNPs. For this, LDH release into the extracellular space was monitored by the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI). The method involves placing cells (2 × 10⁴ cells per mL) in 24-well plates and incubating it with different concentration of Pd-NPs (25–500 μg mL⁻¹) for three and six hours. The plates were then centrifuged, and aliquots (50 μL) of cell culture medium were collected from each well and placed in new microtiter plates. Finally, 50 μL of substrate solution was added to each well and the plates incubated for 30 minutes at room temperature. The absorbance at 490 nm as measured with a microplate reader. Each experiment was done in triplicate. Cytotoxicity is expressed relative to the basal lactate dehydrogenase release by untreated control cells.