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).