Real-time imaging to quantify NETosis with SIEVEWELL
HL-60 cells were cultured for 3 days with 1.3% DMSO to induce differentiation. Neutrophil-differentiated HL-60 cells were incubated for 5 minutes with the membrane-permeable NUCLEAR-ID Red DNA dye to stain nuclei. After three times of washing, NUCLEAR-ID Red stained-neutrophils were loaded into SIEVEWELL. RPMI containing Sytox Green (0.2 µM) and 100 ng/mL PMA was added to induce NETosis and to assess for cell death. SIEVEWELL was placed on IncuCyte® S3 System which is housed inside a cell incubator at 37°C with 5% CO2. Neutrophils were imaged using phase contrast, red (800 ms exposure) and green (400 ms exposure) channels. Images using a 20x dry objective lens were taken every 5 minutes.
Reference of reagents, scan conditions. J Immunol January 15, 2018, 200 (2) 869-879.