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…
Declarações
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
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).