Live-Dead Cell Imaging Assay

The viability of the cells was assessed on days 3, 7 and 21 using a live-dead imaging kit (Molecular Probes, Thermo Fisher Scientific, Hemel Hempstead, UK). As per the manufacturer’s guidelines, the cells underwent incubation with live-dead solution containing 0.05% of 4 mM Cacein- AM (Ex/Em: 495/515 nm) and 0.2% of 2 mM Ethidium homodimer-1 (Ex/Em 495/635 nm) at room temperature for 30 min prior to imaging them with an EVOS fluorescence inverted microscope (EVOS FL color, Life Technologies, Carlsbad, CA, US). The Live/Dead Cell Double Staining Kit is utilised for simultaneous fluorescence staining of viable and dead cells. This kit contains calcein-AM and ethidium solutions, which stain viable and dead cells, respectively. Calcein-AM, an acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though calcein-AM itself is not a fluorescent molecule, the calcein generated from calcein-AM by esterase in a viable cell emits a strong green fluorescence (λex 490 nm, λem 515 nm). Therefore, calcein-AM only stains viable cells. Alternatively, the nuclei staining dye ethidium cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane and intercalates with the DNA double helix of the cell to emit red fluorescence (λex 535 nm, λem 617 nm). Since both calcein and ethidium-DNA can be excited with 490 nm light, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope. The percentage of live and dead cells was calculated after staining.