Cell counts and viability were determined using either trypan-blue staining and manual counting or using the Luna FL automated cell counter (Logos Biosystems, France). Percentage cell viability was calculated by dividing live cells over the total cell count.
Apoptosis was measured using the CellEvent Caspase-3/7 green detection reagent (ThermoFisher Scientific, United Kingdom) according to manufacturer’s instructions. Pelleted cells (20,000) were suspended in reagent and incubated for 45 min at room temperature. Counterstaining was performed with Hoechst for 1 min after which cells were transferred into a cytofunnel (ThermoFisher Scientific) and spun onto a microscope slide using the Cytospin 4 (ThermoFisher Scientific) at 20,000 g for 8 min. Slides were air-dried and mounted using Mowiol aqueous mounting media. Images were taken with a Nikon Eclipse 80i fluorescent microscope at ×40 magnification. For each treatment, green fluorescent cells were considered positive for activated caspase-3/7. For each slide, the total number of caspase positive cells in ten representative fields of view were recorded and calculated as a percentage of the total cells (positive and negative).
Apoptosis was measured using the CellEvent Caspase-3/7 green detection reagent (ThermoFisher Scientific, United Kingdom) according to manufacturer’s instructions. Pelleted cells (20,000) were suspended in reagent and incubated for 45 min at room temperature. Counterstaining was performed with Hoechst for 1 min after which cells were transferred into a cytofunnel (ThermoFisher Scientific) and spun onto a microscope slide using the Cytospin 4 (ThermoFisher Scientific) at 20,000 g for 8 min. Slides were air-dried and mounted using Mowiol aqueous mounting media. Images were taken with a Nikon Eclipse 80i fluorescent microscope at ×40 magnification. For each treatment, green fluorescent cells were considered positive for activated caspase-3/7. For each slide, the total number of caspase positive cells in ten representative fields of view were recorded and calculated as a percentage of the total cells (positive and negative).
Free full text: Click here
