Apr 24, 2026

Flow cytometry to quantify tdTomato+ mDANs

 Forked from Flow cytometry for TH-positive iPSC derived neurons
DOI
https://dx.doi.org/10.17504/protocols.io.kxygx42y4l8j/v1
  • Ellen Hertz1,
  • Gani Perez1,
  • Martha Kirby1,
  • Stacie Anderson1,
  • Yu Chen1,
  • Ellen Sidransky1
  • 1National Institutes of Health
Yu Chen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.kxygx42y4l8j/v1
Protocol Citation: Ellen Hertz, Gani Perez, Martha Kirby, Stacie Anderson, Yu Chen, Ellen Sidransky 2026. Flow cytometry to quantify tdTomato+ mDANs . protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx42y4l8j/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: October 09, 2025
Last Modified: May 18, 2026
Protocol  Integer ID: 229432
Keywords: ASAPCRN, flow cytometry for th, flow cytometry, flow cytometry procedure, derived neuron, neurons this protocol, positive ipsc, ipsc, mdans this protocol, mdan
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
This protocol details the flow cytometry procedure.
Attachments
Icon for n4ysbzvpx.pdf
n4ysbzvpx.pdf
30KB
Materials
Phosphate buffered saline (PBS) without Ca/Mg Thermo Fisher ScientificCatalog #14190144
MemBrite® Fix Cell Surface Staining KitsBiotiumCatalog #30093
PDS Kit Papain VialWorthington Biochemical CorporationCatalog #LK003176
Deoxyribonuclease IWorthington Biochemical CorporationCatalog #LS006333
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016
Paraformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692
Normal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121
Rabbit Tyrosine HydroxylasePel-FreezCatalog #P40101-150
Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #711-605-152
NucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606

mDANs dissociation
5m
Wash mDANs with DPBS once.
10m
Dissociate Day 35 mDNAs with prewarmed papain with DNAse, Y-27632 and Emricasan for 00:10:00 at 37 °C .

Note: iPSCs can be used as the negative control.
Triturate with 1 mL tip to dissociate mDANs in Neurobasal media with 10% fetal bovine serum.

5m
Pellet mDANs by centrifugation at 400 x g, 00:05:00 .
Resuspend dissociated mDANs in Neurobasal media with 10% fetal bovine serum + 10 uM Y-27632 + 5 uM Emricasan to a concentration of 1 million cells per mL.

1h 5m
Filter mDAN suspension through a strainer tube and keep filtered cell suspension on ice.
Flow cytometry
Use iPSCs to define tdTomato- population and use the threshold to determine tdTomato+ population in mDAN suspension.