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

Protocol tips

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	Add 1mL of calcein. Incubate the cells for 15–20 minutes at room temperature, protected from light

Protocol tips
Add 1mL of calcein. Incubate the cells for 15–20 minutes at room temperature, protected from light

Publication protocol

For flow cytometry, cells were washed once in PBS and resuspended in 1mL of calcein (LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells, Molecular Probes, OR, USA) diluted 1/80 in DMSO and 5μL Propidium Iodide (PI) at 0.5mg/mL. The mixed sample was then incubated for 15–20 minutes at room temperature, protected from light. The cells were analyzed by flow cytometry using 488nm excitation and measuring green fluorescence emission for calcein (530/30 bandpass) and red fluorescence emission for PI (610/20 bandpass) on the BD LSRFortessa™ cell analyzer (BD Biosciences, France). Data were exported and analyzed with the Flowjo software.

For fluorescence microscopy, harvested cells were fixed in 4% paraformaldehyde (PFA) at 4°C for 30min. After a PBS wash, cells were adhered to a microscope fluorescence slide. The slides were mounted with SlowFade Gold antifade mountant with DAPI (Life Technologies, Saint-Aubin, France). Observations were carried out using a BX51 fluorescence microscope (Olympus, Rungis, France) and images acquired using the fluorescence imaging system CellA (Olympus, Rungis, France).

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