LDH Assay for Testing Cell Cytotoxicity

The leakage of LDH, a stable cytosolic enzyme, from cells after membrane damage was determined using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, United States). In this enzymatic assay, the enzyme released into culture supernatants converse a tetrazolium salt (iodonitrotetrazolium violet; INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells.

The cells were seeded in a 96-well plate at a density of 1×10⁴ cells per well in 100 μl of the appropriate culture medium for 24 h at 37°C and 5% CO₂ atmosphere. The medium was replaced with fresh medium containing various concentration of AgNPs (1, 2, 3, 4, 5, 10, 16, 32, and 64 μg ml⁻¹). After cell stimulation, 50 μl aliquots of supernatants were transferred to the new wells of 96-well plate and mixed with equal amounts of freshly prepared CytoTox 96 Reagent (Promega, Madison, WI, United States). The microtiter plate was incubated for 30 min at room temperature in the darkness and absorbance measured at 490 nm using the Synergy HT Multi-Mode Microplate Reader (BioTek; Winooski, VT, United States). The AgNP-mediated cytotoxicity was calculated according to the formula:

Culture media backgrounds containing AgNPs concentration were subtracted from the corresponding wells with cells. Blank wells contained the corresponding AgNPs concentration in culture medium. The positive control wells contained cells treated with 0.8% Triton X-100 solution (provided by the manufacturer) for 45 min. These cells released a maximum amount of LDH, which served as 100%.

All obtained data were from three independent experiments with five wells for each experiment. The half inhibitory concentrations (IC₅₀) of AgNPs were determined using GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, United States).