Apr 03, 2023
Live-cell imaging
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi, María José Pérez J. 2023. Live-cell imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjk1xvk5/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: April 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79918
Keywords: ASAPCRN
Abstract
Live-cell imaging is a technique to visualize dynamic cellular processes in living biological samples.
1. Cell Preparation
1. Cell Preparation
Wash cells 1x with OptiMEM (Gibco).
Incubate cells with 100nM MitoTracker red CMH2Xros (Thermo Fisher Scientific) in OptiMEM at 37 °C for 30 min
Wash cells with OptiMEM once, and keep cells in the same medium for imaging
2. Imaging
2. Imaging
Images were acquired using a Leica TCS SP8 confocal microscope (Leica, Germany) with a 100×/1.4 numerical aperture oil-immersion objective.
Analyze images using Diffraction PSF 3D and DeconvolutionLab2 plugins in Fiji-ImageJ version 2.3.0/1.53q (https://fiji.sc; RRID:SCR_002285)
Procedure for labelled alpha-synuclein Pre-formed Fibrils Experiments
Procedure for labelled alpha-synuclein Pre-formed Fibrils Experiments
Treat iPSC-derived neurons with 0.25 μM Alexa Fluor 594-labeled PFFs (594-PFF) with or without cotreatment with 1 μM CDDO-Me (Cayman Chemical)
After 24 h, incubate cells with 100 nM MitoTracker Green (Invitrogen, MA, USA) in a neuronal medium for 30 min at 37 °C
Use ACellBrite™ Steady 488 Membrane Staining Kit (Biotium) to visualize cell membranes following the manufacturer's instructions
Images were acquired using a Leica TCS SP8 confocal microscope with a 63 × /1.4 numerical aperture oil-immersion objective
Acquire Z-stacks for the calculation of the PFF particle area and fluorescence intensity
For each condition, 5-8 images were acquired from at least four independent experiments
Image processing
Image processing
For the quantification of colocalization and image processing, images were analyzed using the “Analyze particles” and “EzColocalization” plugins in Fiji-ImageJ