Jan 28, 2026
Cytotoxicity BioAssay of human iPSC-derived Dopamine Neurons (DANs)
- Humaira Noor1,2,3,4,
- Kaitlyn Cramb5,2,3,4,
- Richard Wade-Martins5,2,3,4
- 1Nufflied Department of Medicine, Henry Wellcome Building for Molecular Physiology, Old Road, University of Oxford, Oxford OX3 7B, UK;
- 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, UK;
- 3Oxford Parkinson’s Disease Centre, University of Oxford, Oxford OX1 3PT, UK;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
- 5Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Sherrington Rd, Oxford, OX1 3PT, UK
Protocol Citation: Humaira Noor, Kaitlyn Cramb, Richard Wade-Martins 2026. Cytotoxicity BioAssay of human iPSC-derived Dopamine Neurons (DANs). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9wdxv4o/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: February 05, 2025
Last Modified: January 30, 2026
Protocol Integer ID: 119616
Keywords: Dopaminergic neurons, iPSCs, differentiation, cytolysis assay, measures adenylate kinase, cytotoxicity bioassay, adenylate kinase, kinetic analysis of cell death, cell lysis of ipsc, derived dopamine neuron, ak in cell culture supernatant, cell death, dopamine neuron, damaged cell, cell culture supernatant, human ipsc, ipsc
Funders Acknowledgements:
Aligning Science Across Parkinson’s Collaborative Research Network
Grant ID: ASAP-020370, ASAP-025192
Abstract
This protocol details a sensitive, fast, high-throughput and non-destructive cytolysis assay that measures adenylate kinase (AK) release from damaged cells. It detects AK in cell culture supernatants, providing real-time, kinetic analysis of cell death without requiring cell lysis of iPSCs-derived DANs plated in a 96-well format plate.
Materials
Reagents:
- Phosphate-buffered saline (PBS), pH 7.4 (Life Technologies, CAT# 10010056)
- Triton X-100 (Sigma- Aldrich, SKU# X100)
- AK assay buffer
Equipment:
- ToxiLight™ Non-Destructive Cytotoxicity BioAssay Kit (Lonza Biologics PLC, CAT# LT07-217)
- Lumitrac™ White 384 well Microplate (Greiner Bio-One Ltd, CAT# 781075)
- PHERAstar® microplate reader (BMG Labtech)
SOLUTIONS
Prepararing 20% Triton X-100 solution:
1. Add 2 mL of Triton X-100 solution in 10 mL PBS.
Prepararing Detection Reagent:
1. Reconstitute the lyophilised AK detection reagent by adding 10 mL of AK assay buffer.
2. Allow it to sit for 15 mins before use.
Troubleshooting
Before start
Cells should be plated at a density of 50 000 cells/ well in a 96 full area plate which has been precoated with Geltrex.
Additionally, care should be taken when adding and removing liquid as these adherent cells are prone to peeling.
Avoid exposure to light by covering the plate with aluminium foil.
Record the record within 5 minutes of adding the reagent to the supernatant.
Collect 5 μl of cell supernatant and transfer to a 384-well Greiner LUMITRAC™ plate.
Add 25 μl of detection reagent to each well and keep it for 5 minutes.
Record luminescence was using a PHERAstar® microplate reader.
For positive control, add an equal volume of 20% Triton X-100 solution to the media of one well per cell line for 5 minutes before supernatant collection.