DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation

Abstract

Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs). J. Vis. Exp. (2018)

In this video, we determine the proliferative capacity of neuronal progenitor cells by measuring the incorporation of radioactive tracer tritiated thymidine into the DNA of cells. This technique can be used to study cell proliferation after treatment with certain drugs and growth factors.

Protocol

1. Maintenance of NPCs

1. Plate NPCs at a density of 1.5 million cells into an ECM-mimic gel-coated well containing 2 mL of 100% Expansion Media.
2. Incubate NPCs at 37 °C in a moist environment with 5% CO₂.
3. Add 5 μM of ROCK inhibitor to the media for NPCs at P3 or lower, or for thawed NPCs, to prevent excessive cell death. Change media after 24 h to remove ROCK inhibitor.
4. Passage cells every 4–9 days, depending upon when they become confluent. Cells are considered confluent when they become a densely packed monolayer covering the entire bottom of the dish surface.
5. Plate NPCs at ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM/F12 Medium	ThermoFischer Scientific	12634-010	Component of 100% Expansion Medium
Neurobasal Medium	ThermoFischer Scientific	21103049
24-well plates	ThermoFischer Scientific	2021-05	Polystyrene plates: Used for NPC DNA Synthesis Assay
Natural Mouse Laminin	Invitrogen	23017-015	Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays
Penicillin/Streptomycin	ThermoFischer Scientific	15140122	Antibiotic, component in Expansion Media
2.5% Trypsin (10X)	Gibco	15090-046	10X enzymatic solution
0.5 M EDTA	ThermoFischer Scientific	AM9261	Used in trypsin solution for lifting cells for DNA Synthesis Assay
tritiated [3H]-thymidine	PerkinElmer	NET027E001	Radioactive tritium, thymidine
Fisherbrand 7 mL HDPE Scintillation Vials	Fisherbrand	03-337-1	Vials for liquid scintillation counting
EcoLite(+)	MP Biomedicals	188247501	Liquid scintillation cocktail
LS 6500 multi-purpose liquid scintillation counter	Beckman Coulter	8043-30-1194	Liquid Scintillation Counter
Skatron Semi-automactic Cell Harvester	Molecular Devices & Skatron Instruments, Inc.	Type 11019	Semi-automatic cell harvester
Grade GF/C filter paper, Whatman	GE Healthcare Life Sciences	1822-849	Glass fiber filter paper