Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Prepare a staining solution of 2 uM calcein AM/4 uM EthD-III by adding 5 uL of 4 mM calcein AM and 20 uL of 2 mM EthD-III to 10 mL of PBS or other serum-free buffer or medium	Wash cells before adding calcein AM/EthD-III staining solution to cover the cell monolayer. Incubate the cells for 30-45 minutes at room temperature.

Upstream tips
Prepare a staining solution of 2 uM calcein AM/4 uM EthD-III by adding 5 uL of 4 mM calcein AM and 20 uL of 2 mM EthD-III to 10 mL of PBS or other serum-free buffer or medium
Protocol tips
Wash cells before adding calcein AM/EthD-III staining solution to cover the cell monolayer. Incubate the cells for 30-45 minutes at room temperature.

Publication protocol

After spheroid contraction for 72 h, fluorescent staining was performed to assess viability and cytoskeletal organization within the spheroids. Throughout staining, spheroids were anchored to the bottom of the well plate using the 96-well magnetic drive. Spheroids stained for viability (Live/Dead, Biotium, Hayward, CA) were first gently washed with PBS using a multichannel pipet, after which, a solution containing 2 μM calcein AM and 4 μM ethidium homodimer-III was added to each well to incubate for 30 min. The spheroids were then washed again with PBS and imaged under a fluorescent microscope.

Spheroids stained for cytoskeletal organization were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for at least 5 h. After washing twice with PBS, a solution containing fluorescently-tagged phalloidin (AlexaFluor, Invitrogen, Carlsbad, CA) in PBS was added to each well for 30 min to stain for F-actin. The spheroids were then washed with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI, KPL, Gaithersburg, MD) for 15 min. After one final wash, the spheroids were imaged under a fluorescent microscope.

Spheroids were also stained for nuclei to visualize contraction after printing. Spheroids were printed and fixed with 4% paraformaldehyde 0, 1, 3, and 6 h after printing for at least 5 h. After washing twice in PBS, the spheroids were stained for nuclei with DAPI for 15 min, then imaged under a fluorescent microscope.

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Manufacturer protocol

Download the product protocol from Biotium for Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells below.

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