LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells

Protocol tips

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	Add ethidium-calcein mixture, and incubate for 30 min in a 37 °C incubator

Protocol tips
Add ethidium-calcein mixture, and incubate for 30 min in a 37 °C incubator

Publication protocol

For live imaging, spheroids of MRC-5 cell line were stained for DNA with Hoechst 33342 (Molecular Probes™). In particular, we exposed the spheroids to a solution of PBS 1x supplemented with Hoechst 33342 1 μg/ml. Viability of the A549 spheroids was measured with the LIVE/DEAD® Cell Viability Assay (Molecular Probes™). Briefly, samples were washed with warm phosphate-buffered saline (PBS), added to an ethidium-calcein mixture, and incubated for 30 min in a 37 °C incubator. After washing again with PBS, the spheroids were ready for the imaging procedure. All spheroids were mixed with low-melt agarose solution (Carl Roth GmbH) and the mixture was sucked into glass capillaries, with inner diameter of 1 mm. The agarose was allowed to gel at room temperature for five minutes before imaging.

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