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 a density of 1 to 1.5 million cells per one well of a 6-well plate. Remove spent media every 48 h and replace with 2 mL of 100% Expansion Media.

3. Neural Precursor Cell DNA Synthesis Assay

1. Plate 100,000 cells/well in a 24-well plate and assess in triplicate/condition.
2. Add radioactive, tritiated [³H]-thymidine to the culture medium (1.5 μCi/mL) in each well after 46 h in culture. Incubate cells at 37 °C for 2 h.
   NOTE: When using radioactive materials, receive training from your institution, follow radioactivity safety protocols, and dispose of radioactive materials in the appropriately designated waste receptacles.
3. Remove and properly dispose of radioactive media after 2 h. Add 300 μL of pre-warmed 0.25% trypsin-EDTA (0.5 mM) to each well and incubate for 20 min at 37 °C.
4. Turn on the cell harvester (see Materials section) and the pump and ensure that the pump pressure is below 200 PSI. Place filter paper through the space in the cell harvester and tear off the right corner to mark paper orientation.
5. Place collecting tubes of cell harvester into an empty "blank" tray and press prewash to moisten the filter paper. Place collecting tubes into sample wells and run (start).
6. When the cell harvester has finished collecting samples, lift clamp and advance filter paper to repeat for all sample sets. Always prewash in an empty, "blank" plate.
7. Dry filter paper under a light source and set up corresponding vials in a tray. Punch out paper chads into vials and add 2 mL of liquid scintillation cocktail to each vial. Cap and label vials.
8. Incubate vials in a liquid scintillation cocktail for at least 1 h before reading the counts per minute (CPMs) on a scintillation machine.

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