LIVE/DEAD™ Cell Imaging Kit

Protocol tips

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	Incubate for 15 minutes at 20–25°C

Protocol tips
Incubate for 15 minutes at 20–25°C

Publication protocol

Peritoneal macrophages and RAW cells were infected with the indicated dose of LM for 24h. For the lactate dehydrogenase (LDH) release assay, culture supernatant was collected after infection and cell death was quantified using a cytotoxicity detection kit according to the manufacturer’s instructions (Roche). For the cell imaging assay, cells were stained with a LIVE/DEAD Cell Imaging kit, according to the manufacturer’s instructions (Molecular Probes) and imaged on Keyence BZ-9000 to determine whether they were live (green) or dead (red). Using an MEBCYTO Apoptosis Kit (MBL), cells were washed in PBS and re-suspended in 100 μl of binding buffer. Cells were then incubated with 10 μl of annexin V-FITC for 15min at room temperature in the dark, followed by the addition of 400 μl of binding buffer and analysis using a BD FACSCanto II. For the cytotoxicity assay, cells were seeded 24h before the assay in 96-well plates at a density of 2×105 cells per well. Cells were treated with the indicated dose of pyocyanin in the presence or absence of AIM. After treatment for 24h, cell viability was assessed with a Cell Counting Kit (Dojin Laboratories, Kumamoto, Japan).

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