Cellstain-Double Staining Kit

Protocol tips

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Prepare a mix by adding 2 µL of 1 M calcein-AM stock solution and 3 µL of 1.5 M PI solution to 1 mL of Dulbecco’s modified Eagle’s medium supplemented with 2 mM of l-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin.	Mix 100 µl of the staining solution with 200 µl of the cell suspension, and incubate the mixture for 15 minutes at 37C

Upstream tips
Prepare a mix by adding 2 µL of 1 M calcein-AM stock solution and 3 µL of 1.5 M PI solution to 1 mL of Dulbecco’s modified Eagle’s medium supplemented with 2 mM of l-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin.
Protocol tips
Mix 100 µl of the staining solution with 200 µl of the cell suspension, and incubate the mixture for 15 minutes at 37C

Publication protocol

Calcein-AM and propidium iodide (PI) were used to identify live and dead cells, respectively (Cellstain Double Staining Kit; DOJINDO, Kumamoto, Japan). The assay solution mixing calcein-AM and PI was prepared by adding 2 µL of 1 M calcein-AM stock solution and 3 µL of 1.5 M PI solution to 1 mL of Dulbecco’s modified Eagle’s medium supplemented with 2 mM of l-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin. The cells on TRCD and the cell sheets after two and 24 hours of reculturing on a glass-based dish (IWAKI, Asahi Glass Co., LTD., Tokyo, Japan) were incubated with the assay solution for 15 minutes and then washed with PBS solution. The double-stained cells were assessed using a fluorescence microscope (Eclipse Ti-U; Nikon).

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