Sep 23, 2025

Public workspaceFlow cytometry for TH-positive iPSC derived neurons

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbxwkqlpk/v1
  • Ellen Hertz1,
  • Ellen Hertz1,
  • Gani Perez1,
  • Martha Kirby1,
  • Stacie Anderson1,
  • Yu Chen1,
  • Ellen Sidransky1
  • 1National Institutes of Health
Ellen Hertz
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.81wgbxwkqlpk/v1
Protocol Citation: Ellen Hertz, 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: September 23, 2025
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.
Attachments
Icon for n4ysbzvpx.pdf
n4ysbzvpx.pdf
30KB
Materials
ReagentPhosphate buffered saline (PBS) without Ca/Mg Thermo Fisher ScientificCatalog #14190144
ReagentMemBrite® Fix Cell Surface Staining KitsBiotiumCatalog #30093
ReagentPDS Kit Papain VialWorthington Biochemical CorporationCatalog #LK003176
ReagentDeoxyribonuclease IWorthington Biochemical CorporationCatalog #LS006333
ReagentDMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016
ReagentParaformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692
ReagentNormal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121
ReagentRabbit Tyrosine HydroxylasePel-FreezCatalog #P40101-150
ReagentAlexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #711-605-152
ReagentNucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606

Troubleshooting
Flow cytometry dopaminergic fraction
5m
Wash the neurons in PBS and incubate with Membrite prestain solution, dilute 1:1000 in HBSS, for Duration00:05:00 at Temperature37 °C .
5m
Incubation
Wash
Change the prestain solution to staining solution and incubate for another Duration00:05:00 at Temperature37 °C .

5m
Incubation
Dissociate the cells with prewarmed papain with DNAse, Y-27632 and Emricasan for Duration00:10:00 at Temperature37 °C .
10m
Quench the reaction in DMEM/Glutamax with 10% fetal bovine serum and collect by centrifugation at Centrifigation400 x g, 00:05:00 .

5m
Centrifigation
Resuspend the pellet in 4% paraformaldehyde and incubate for Duration00:10:00 at TemperatureRoom temperature on a rolling wheel.

10m
Incubation
Resuspend the cells in DMEM with 10% FBS and spin at Centrifigation500 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 Duration00:30:00 at TemperatureRoom 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) DurationOvernight at Temperature4 °C .

1h 5m
Centrifigation
Overnight
Wash the cells twice with 2% BSA and Concentration1.5 millimolar (mM) EDTA in PBS and then incubate with diluted Alexa647-conjugated secondary antibody, for Duration01:00:00 at TemperatureRoom temperature .

1h
Incubation
Wash
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.
Wash
Initial gate: Include Membrite+ cells
Second gate: Include single cell DAPI (exclude duplicates).
Third gate: Alexa647+