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

Published: April 30, 2023

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 Plate NPCs at a density of 1.5 million cells into an ECM-mimic gel-coated well containing 2 mL of 100% Expansion Media. Incubate NPCs at 37 °C in a moist environment with 5% CO2. 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. Passage cells every 4–9 days, depending upon when they become confluen…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation. J. Vis. Exp. (Pending Publication), e20629, doi: (2023).

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