Live-cell imaging; cell death assay
Minee L Choi
Abstract
This protocol contains instructions for examining cell death in live-cell imaging.
Steps
Cell death is detected using Propidium iodide (PI, Thermo Fisher Scientific) or SYTOX™ Green (SYTOX, Thermo Fisher Scientific) which is excluded from viable cells but exhibits red (PI) and green (SYTOX) fluorescence following a loss of membrane integrity and Hoechst 33342 (Hoechst, Thermo Fisher Scientific).
20 μM PI or 500 nM SYTOX and 10 uM Hoechst are directly added to the dishes.
Cells are incubated for 15 min.
Live-cell imaging is performed using a confocal microscope.
Hoechst and PI are excited by 405 nm and 543 nm laser lines with the emission between 405 nm to 470 nm and 570 nm to 640nm respectively.
SYTOX is excited by a 488 nm laser with emissions between 488 nm and 516 nm.
