Sep 23, 2025

Flow cytometry for TH-positive iPSC derived neurons

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbxwkqlpk/v1
  • Ellen Hertz1,
  • Gani Perez1,
  • Martha Kirby1,
  • Stacie Anderson1,
  • Yu Chen1,
  • Ellen Sidransky1
  • 1National Institutes of Health
Ellen Hertz
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbxwkqlpk/v1
Protocol Citation: Ellen Hertz, Gani Perez, Martha Kirby, Stacie Anderson, Yu Chen, Ellen Sidransky 2025. Flow cytometry for TH-positive iPSC derived neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxwkqlpk/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: November 21, 2023
Last Modified: May 18, 2026
Protocol  Integer ID: 92533
Keywords: ASAPCRN, flow cytometry for th, flow cytometry, flow cytometry procedure, derived neuron, neurons this protocol, positive ipsc, ipsc
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
This protocol details the flow cytometry procedure.
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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

Flow cytometry dopaminergic fraction
5m
Wash the neurons in PBS and incubate with Membrite prestain solution, dilute 1:1000 in HBSS, for 00:05:00 at 37 °C .
5m
Change the prestain solution to staining solution and incubate for another 00:05:00 at 37 °C .

5m
Dissociate the cells with prewarmed papain with DNAse, Y-27632 and Emricasan for 00:10:00 at 37 °C .
10m
Quench the reaction in DMEM/Glutamax with 10% fetal bovine serum and collect by centrifugation at 400 x g, 00:05:00 .

5m
Resuspend the pellet in 4% paraformaldehyde and incubate for 00:10:00 at Room temperature on a rolling wheel.

10m
Resuspend the cells in DMEM with 10% FBS and spin at 500 x g, 00:05:00 after which the cells were incubated in permeabilization buffer (1% BSA, 0.1% saponin in PBS, 10% normal donkey serum for 00:30:00 at Room temperature and thereafter label with tyrosine hydroxylase antibody or isotype controls (1:400, Cat.# P40101-150, Pel-Freeze and Cat.# AB-105-C R&D respectively) Overnight at 4 °C .

1h 5m
Wash the cells twice with 2% BSA and 1.5 millimolar (mM) EDTA in PBS and then incubate with diluted Alexa647-conjugated secondary antibody, for 01:00:00 at Room temperature .

1h
Wash the cells as described above with NucBlue Fixed Cell ReadyProbes Reagent in the second wash and resuspend in wash buffer for flow cytometry analysis.
Initial gate: Include Membrite+ cells
Second gate: Include single cell DAPI (exclude duplicates).
Third gate: Alexa647+