Jul 23, 2025

Public workspaceStimulation of DA neurons and microglia

DOI
https://dx.doi.org/10.17504/protocols.io.261geko2dg47/v1
  • Arun Thiruvalluvan1,2,
  • Agnete Kirkeby1,2
  • 1Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.;
  • 2Novo Nordisk Foundation Center for Stem Cell Medicine (renew), University of Copenhagen, 2200 Copenhagen, Denmark
Laura Castilla-Vallmanya
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DOI: https://dx.doi.org/10.17504/protocols.io.261geko2dg47/v1
Protocol Citation: Arun Thiruvalluvan, Agnete Kirkeby 2025. Stimulation of DA neurons and microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.261geko2dg47/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: July 16, 2025
Last Modified: July 23, 2025
Protocol Integer ID: 222630
Keywords: ASAPCRN, microglia this procotol, microglia cultures with ifn, microglia culture, cells for bulkrna, microglia, stimulation of da neuron, procotol, bulkrna, dopaminergic neuron, da neuron, neuron, cell, details for stimulation, stimulation
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-024296
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This procotol describes the details for stimulation of dopaminergic neurons and microglia cultures with IFN-gamma, TNF-alpha and Poly-IC and how to harvest the cells for bulkRNA-seq.
Troubleshooting
Stimulation
2d
Terminally differentiated DA neurons and microglia were stimulated with different compounds diluted in the appropriate culture medium for Duration48:00:00 at Temperature37 °C

CompoundConcentrationProviderCat# Number
IFN-gamma5ng/mlMiltenyi Biotec130-096-873
TNF-alfa5ng/mlMiltenyi Biotec130-094-014
Poly IC10ng/mlSigma AldrichP1530-25MG

2d
Harvesting for bulk-RNAseq
Post stimulation, cells were washed twice with 1XPBS and detached with Accutase (Thermo Fisher Scientific: A1110501). Add 500ul/well of accutase and incubate for 7-8min (see under the microscope, if all the cells are detached or leave longer until they detach)
Collect samples in the labelled tubes and wash the plate again with PBS to collect remaining cells in the well, and add them to the same tube.
Spin down the tube containing samples at 4000 rpm for 5 minutes. Remove the supernatant and place the pellet on dry ice.
Cell pellets were frozen down on dry ice and kept at Temperature-80 °C for bulk-RNA seq.