Nov 16, 2025

Public workspaceCalcium Imaging Protocol for hIPSC-derived Dopaminergic neurons

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld65d7g5b/v1
  • Judith Kreutzmann1
  • 1Karolinska Institute
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld65d7g5b/v1
Protocol Citation: Judith Kreutzmann 2025. Calcium Imaging Protocol for hIPSC-derived Dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld65d7g5b/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 09, 2025
Last Modified: November 16, 2025
Protocol Integer ID: 226859
Keywords: calcium imaging protocol for hipsc, calcium imaging protocol, imaging protocol to analyse calcium dynamic, dopaminergic neuron, derived dopaminergic neuron, analyse calcium dynamic, imaging protocol
Abstract
This is the imaging protocol to analyse calcium dynamics in vitro KOLF2.1J differentiated midbrain dopaminergic neurons
Protocol materials
ReagentFluo-4 Calcium Imaging KitThermo FisherCatalog #F10489
ReagentPluronic® F-127Merck MilliporeSigma (Sigma-Aldrich)Catalog #P2443
Troubleshooting
Preparation of Loading Solution
Prepare the dye-loading solution in Krebs-Ringer buffer:
  • 5 µM Fluo-4 AM ReagentFluo-4 Calcium Imaging KitThermo FisherCatalog #F10489
  • 0.625% Pluronic F-127ReagentPluronic® F-127Merck MilliporeSigma (Sigma-Aldrich)Catalog #P2443
Mix thoroughly to ensure dye solubilization.

Mix
Dye Loading
30m
Add loading solution to the 35 mm culture dishes containing live cells.
Incubate at 37°C for 30 minutes in the dark. Temperature37 °C

30m
Washing
After incubation, wash cells twice with fresh Krebs-Ringer buffer to remove excess dye.
Maintain cells in fresh Krebs-Ringer buffer at 37°C during imaging. Temperature37 °C
Calcium Imaging
Perform imaging using a Nikon CrEST X-Light V3 spinning disk confocal microscope:
  • Objective: 20x Air lens
  • Excitation wavelength: 480 nm
  • Sampling frequency: 1 Hz (one frame per second)
Use Nikon NIS-Elements software to control the microscope and acquire time-lapse fluorescence data.
Imaging
Data Analysis
Export image sequences from NIS-Elements.

Analyze calcium signals using the following tools:
  • FIJI (ImageJ) for image preprocessing and intensity extraction.
Software
ImageJ/Fiji
NAME
Windows 7
OS
National Institutes of Health
DEVELOPER
http://wsr.imagej.net/distros/win/ij152-win-java8.zip
REPOSITORY
https://imagej.nih.gov/ij/
SOURCE LINK

  • MATLAB (R2020b) for custom analysis scripts.
Software
MATLAB
NAME
Microsoft Windows 10
OS
Mathworks
DEVELOPER
Company Website
REPOSITORY
https://www.mathworks.com/products/matlab.html
SOURCE LINK

  • FluoroSNNAP (GitHub: https://github.com/tapan-patel/FluoroSNNAP) for spike detection and activity analysis.