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

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Wash cells 2–3 times with PBS	Add calcein AM: 2 μM; EthD-1: 4 μM Incubate the cells for 40 minutes at room temperature

Upstream tips
Wash cells 2–3 times with PBS
Protocol tips
Add calcein AM: 2 μM; EthD-1: 4 μM Incubate the cells for 40 minutes at room temperature

Publication protocol

After NP cells (seeded in a 6-well plate, 5 × 103 cells/well) were incubated with different test compounds for 1, 3 and 5 days, cell viability was assessed using a LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen, USA). Briefly, after washing the cells 2–3 times with PBS, NP cells were incubated with a fluorescent working solution (calcein AM: 2 μM; EthD-1: 4 μM) for 40 minutes at room temperature. Then, the live (green fluorescence) or dead (red fluorescence) NP cells in each group were viewed under a fluorescence microscope (Olympus IX71, Japan). Cell viability (expressed as the live to total cell ratio) in the TNF-α-incubated groups was quantified relative to the control group (%) using Image-Pro Plus software (Version 5.1, Media Cybernetics, Inc.)

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