[3H]Thymidine Incorporation Assay for Rat-1a Cells
This
method of Peter Coward, Ph.D., was used in Coward, et al (1998) Controlling
signaling with a specifically designed Gi-coupled receptor Proc. Natl. Acad.
Sci. 95:352–357.
- 1. "Making
cells quiescent," i.e. synchronizing the cells in a low growth
state.
- Seed 500,000
cells per well in a 24-well plate in DME + 10% calf serum.
- Incubate 12–24
hours.
- Rinse 1X with
serum-free media.
- Add 1 ml serum-free
media.
- Incubate 24
hours.
- 2. Stimulating
proliferation and labeling with [3H]thymidine
- Add drug. Incubate
16 hours.
- Add 1 micro
curie [3H]thymidine (NEN #NET-027Z) (1 ul diluted with 24 ul of media) to
each well.
- Incubate 8 hours.
- 3. Extraction
of [3H]thymidine labeled DNA
- Aspirate media.
- Wash carefully
with 1 ml ice cold PBS.
- Aspirate PBS.
- Add 1 ml of
ice cold 5% TCA. Leave at 4 deg C for 30 minutes.
- Aspirate and
wash once with PBS.
- At room temperature,
add 0.5 ml 0.5N NaOH/0.5% SDS.
- Pipette up and
down and add to scintillation vials.
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