Live/Dead Cell Double Staining Kit

Protocol tips

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	Add calcein-AM 10 μM and ethidium homodimer 15 μM, in PBS, for 20 min at 37 °C

Protocol tips
Add calcein-AM 10 μM and ethidium homodimer 15 μM, in PBS, for 20 min at 37 °C

Publication protocol

The cell viability test employed to investigate NP toxicity in MW as well as in MFD was Live/Dead cell staining kit (Sigma-Aldrich®) that assesses cell viability by calcein-AM and propidium iodide (PI) fluorescence, which stain viable and dead cells, respectively. Briefly, cells were seeded on a glass coverslip or in a MFD and allowed to attach. After incubation with Au NP (24 h), cell culture medium was removed, and the cells were washed twice in PBS. The cells were then incubated with calcein-AM 10 μM and ethidium homodimer 15 μM, in PBS, for 20 min at 37 °C and washed twice in PBS. Stained cells were then detected by fluorescence microscopy (Leica Microsystems): the calcein generated from calcein-AM by esterase in viable cells emitted green fluorescence (excitation, 490 nm; emission, 515 nm), whereas PI, intercalated with DNA by passing through disordered areas of dead cell membrane, emitted red fluorescence (excitation, 535 nm; emission, 617 nm). For every administered NP concentration, the cell survival rate was measured as the number of living cells expressed as percentage of the number of control cells, which underwent the same processing steps but did not receive NP.

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