Sep 21, 2022
Live-cell imaging: cell death assay
- gurvir.virdi 1
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. Live-cell imaging: cell death assay. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj8yywgk5/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: September 21, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70339
Keywords: ASAPCRN, cell death in midbrain, examining cell death, midbrain dopaminergic neuron, cell death, dopaminergic neuron, cell imaging, neuron, using sytox green, sytox green, cell, midbrain, death, imaging
Abstract
Examining cell death in midbrain dopaminergic neurons using SYTOX Green
Troubleshooting
Cell death is detected using SYTOX™ Green (SYTOX, Thermo Fisher Scientific) which is excluded from viable cells but exhibits green (SYTOX) fluorescence following a loss of membrane integrity.
Hoechst 33342 (Hoechst, Thermo Fisher Scientific) is used as a nuclear cell stain.
Cells were incubated with 500 nM SYTOX and 10 uM Hoechst for 00:40:00 at room temperature made up in HBSS.
40m
Live-cell imaging is then performed using a confocal microscope.
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 1% of laser output to prevent phototoxicity, and the pinhole is set to allow optical slice at approximately 1-2 μm.
Hoechst is excited by 405 nm laser line with the emission between 405 nm to 470 nm.
SYTOX is excited by a 488 nm laser with emissions between 488 nm and 516 nm.