Keratinocytes were incubated in media containing 50 nM sytox orange (ThermoFischer #S11368) and 0.2 µg/mL Hoechst 34342 (Sigma #H3570). Cells were stimulated and imaged over time using the Biotek Cytation 5 Cell Imaging Multi-Mode Reader with fluorescent DAPI and RFP filter cubes at 37 °C with 5% CO2. Cellular analysis was performed with built-in Gen5 Cytation 5 software and DAPI/RFP double-positive cells were quantified with built-in software.
Protocol detail
Quantifying Apoptosis in Keratinocytes
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Corresponding Organization :
Other organizations : University of Massachusetts Chan Medical School, University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Stimulation of cells
- Cell viability/death (Sytox orange-positive cells)
- Cell number (Hoechst 34342-positive cells)
- Incubation media composition (50 nM Sytox orange and 0.2 µg/mL Hoechst 34342)
- Imaging conditions (37 °C, 5% CO2, fluorescent DAPI and RFP filter cubes)
- Cell type (Keratinocytes)
- Positive control: Not specified
- Negative control: Not specified
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