Feb 03, 2025
Functional assays via live‒cell imaging
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Functional assays via live‒cell imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wkn8vo5/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112817
Funders Acknowledgements:
ASAP
Abstract
Functional assays via live‒cell imaging
Functional assays via live‒cell imaging
Functional assays via live‒cell imaging
Incubate cells with specific fluorescent dyes tailored to each desired measurement for 30 minutes at 37°C.
Wash cells twice with PBS
Conduct imaging using a Leica TCS SP8 confocal microscope equipped with a 63×/1.4 numerical aperture oil immersion objective
Perform all measurements and analyses on data obtained from over 40 cells across three independent experiments
For mitochondrial function assessment
For mitochondrial function assessment
Use 100 nM tetramethylrhodamine methyl ester perchlorate (TMRM) and 100 nM MitoTracker Green (Invitrogen)
Use an excitation wavelength of 568 nm for TMRM and 488 nm for MitoTracker Green
Measure emitted fluorescence at wavelengths shorter than 574 nm for TMRM and between 490-530 nm for MitoTracker Green
Quantify TMRM intensity in mitochondria stained with MitoTracker Green using Fiji (version 2.7.0, RRID: SCR_002285
Set basal TMRM fluorescence intensity at 100% and determine background fluorescence after mitochondrial depolarization with FCCP. Subtract the background value from the basal value for corrected intensity
For calcium measurements
For calcium measurements
Incubate iPSC-derived neurons and astrocytes with 1 µM Fluo-4 AM (Invitrogen)
Use an excitation wavelength of 488 nm and emission range of 490-530 nm for Fluo-4 AM
Acquire images every 25 seconds
Stimulate neurons with 0.6 mM KCl and astrocytes with 1 mM glutamate for 15 minutes following a 1-minute equilibration period to establish baseline fluorescence intensity
For oxidative stress evaluation
For oxidative stress evaluation
Incubate cells with 1 µM DCF (Invitrogen)
Use an excitation wavelength of 488 nm and emission range of 490-530 nm
Measure fluorescence intensity using Fiji (version 2.7.0, RRID: SCR_002285)
Normalize fluorescence intensity values to baseline values.