Feb 01, 2024
Live-cell imaging; cell death assay
- 1UCL Institute of Neurology
Protocol Citation: Minee L Choi 2024. Live-cell imaging; cell death assay. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy1d5lx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 26, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63250
Keywords: ASAPCRN
Abstract
This protocol contains instructions for examining cell death in live-cell imaging.
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.
Note
Live-cell imaging is performed using a confocal microscope (Zeiss LSM 710 or 880 with an integrated META detection system). For confocal microscopes, illumination intensity is limited to 0.1-0.2% of laser output to prevent phototoxicity, and the pinhole is set to allow optical slice at approximately 1-2 μm.
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.