Overall cell death was evaluated by fluorescent microscopy as previously described (41 (link)) with modifications. Briefly, the cells were cultured on an 8-well imaging chamber (Imaging Chamber 8 CG; Zell-Kontakt GmbH, Nörten-Hardenberg, Germany) and treated with the agents to be tested for 24 h at 37°C. Subsequently, the cells were stained with 4 µM each of calcein-AM and ethidium bromide homodimer-1 (EthD-1) to label live cells in green and dead cells in red, respectively using a commercially available kit (Live/Dead Viability/Cytotoxicity kit; Invitrogen/Thermo Fisher Scientific) according to the manufacturer's instructions. Images were obtained using a BZ X-700 fluorescence microscope (Keyence Corp., Osaka, Japan) equipped with a 40×, 0.60 numerical aperture (NA) LUCPlanFL N objective (Olympus, Tokyo, Japan) and analyzed using BZ-H3A application software (Keyence Corp.).
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Evaluation of Cell Death by Fluorescent Microscopy
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Variable analysis
independent variables
- Agents tested
- Overall cell death
- Cells cultured on an 8-well imaging chamber
- Cells treated for 24 h at 37°C
- Cells stained with 4 µM each of calcein-AM and ethidium bromide homodimer-1 (EthD-1)
- Imaging performed using a BZ X-700 fluorescence microscope with a 40×, 0.60 numerical aperture (NA) LUCPlanFL N objective
- Positive control: Live cells stained with calcein-AM (green fluorescence)
- Negative control: Dead cells stained with ethidium bromide homodimer-1 (red fluorescence)
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