May 16, 2025

Public workspaceIntracellular ROS measurement in iPSC-derived microglia V.1

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxqb9wv8j/v1
  • Irene Fernández-Carasa1,2,
  • Lucas Blasco-Agell1,2,
  • Jara Montero-Muñoz1,2,
  • Antonella Consiglio1,2,
  • Miquel Vila3
  • 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
  • 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain.;
  • 3VHIR-CIBERNED-ASAP
  • Vilalab Public
Miquel Vila
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxqb9wv8j/v1
Protocol Citation: Irene Fernández-Carasa, Lucas Blasco-Agell, Jara Montero-Muñoz, Antonella Consiglio, Miquel Vila 2025. Intracellular ROS measurement in iPSC-derived microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxqb9wv8j/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 16, 2025
Last Modified: May 16, 2025
Protocol Integer ID: 218432
Keywords: microglia intracellular ros measurement in ipsc, derived microglia intracellular ros measurement, intracellular ros measurement in ipsc, intracellular ros measurement, microglia
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
Intracellular ROS measurement in iPSC-derived microglia
Troubleshooting
Intracellular ROS is measured in hMG exposed to 5 ng/mL NM for 24 hours using 2’,7’- Dichlorodihydrofluorescein diacetate (DCFH2-DA) probe (Sigma-Aldrich), which turns highly fluorescent upon oxidation.
Ten minutes before adding the probe, hydrogen peroxide (1:25) is added to a well as a positive control of oxidation and absolute ethanol as negative control.
Then, 500 μM of DCFH2-DA are added for one hour at 37ºC. After that, cells are rinsed and recovered with a scrapper in PBS with 1% Triton.
Samples are placed in CellCarrier‱-96 Ultra Microplate and fluorescence is measured within a FLUOstar Omega microplate reader (BMG LABTECH) at 485nm/530nm.
Results are normalized to protein concentration measured using a Micro BCATM Protein Assay Kit (Thermo Fisher Scientific).