[3H]-Thymidine Incorporation Assay for Rat-1a Cells

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Tony Rook
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[3H]-Thymidine Incorporation Assay for Rat-1a Cells

Please find the following protocol:
 [3H]-Thymidine Incorporation Assay for Rat-1a Cells
Overview:
The rate of proliferation of cells can be measured by determining the incorporation of [3H]-thymidine into cellular nucleic acids.
Procedure:
Synchronizing Cells in a Low Growth State to Make Cells Quiescent
 1. Seed 500,000 cells per well in a 24-well plate in DMEM+ 10% Fetal Calf Serum.
 2. Incubate cells in a 37°C incubator for 12 to 24 hr.
 3. Remove the media from the wells and rinse the cells once with serum-free media (DMEM).
 4. Add 1 ml of serum-free media to each well. 
5. Incubate the cells for 24 hr in a 37°C humidified 5% CO2 incubator.
Stimulating cell proliferation and labeling with [3H]-Thymidine  
1. Add Dynorphin or Spiradoline to a concentration in the range of 0.05 pM to 1 μM.
 2. Incubate cells for 16 hr in a 37°C humidified 5% CO2 incubator.
 3. Add 1 μCi of [3H]-Thymidine (NEN) to each well. For ease of manipulation, add 1  μl of Thymidine (1 μCi) to 24 μl of media . Then add this mixture to each well.
4. Incubate the cells 8 hr in a 37°C humidified 5% CO2 incubator.
Extraction of [3H]-Thymidine-labeled DNA
1. Aspirate the media from each well.  
2. Wash the cells carefully with 1 ml of ice-cold PBS.
 3. Aspirate PBS.
 4. Add 1 ml of ice-cold 5% TCA.
 5. Incubate cells at 4°C for 30 min.
 6. Aspirate TCA solution and wash once with PBS.
 7. At room temperature, add 0.5 ml of 0.5 M NaOH/0.5% SDS Solution.
 8. Pipette the cell suspension up and down to lyse cells and add this mixture directly to scintillation vials containing Scintillation Fluid.
 9. Count the vials in a scintillation counter for assessment of radioactive nucleotide incorporation.
Solutions:
 
PBS.............................................................................pH 7.2
 
.......................................................................2.7 mM KCl
 
.....................4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
 
...............1.8 mM Potassium Phosphate Monobasic (KH2PO4)
 
....................................................................137 mM NaCl
 
70-90 Ci/mmole methyl-[3H]-Thymidine (NEN, 1 mCi/ml) ...............................................CAUTION! Biohazard
 
DMEM (Dulbecco's Modified Eagle's Medium) + 10% Fetal Calf Serum
 0.5 M NaOH/0.5% SDS Solution.......................0.5% (w/v) SDS
 ..............................0.5 M NaOH
 5% (w/v) TCA................................CAUTION! Biohazard
 
Bioreagents and Chemicals:
 
Sodium Phosphate, Dibasic
 Potassium Phosphate, Monobasic
 Potassium Chloride
 Sodium Chloride
 Sodium Hydroxide
 SDS
 Trichloroacetic Acid (TCA)
 Scintillation Fluid
 [3H]-Thymidine
 Fetal Calf Serum (FCS) 
DMEM
 Spiradoline
 Dynorphin
Reference and Links:
 1. Coward, et al. Controlling signaling with a specifically designed Gi-coupled receptor. PNAS 1998; 95:352-357.

http://openwetware.org/wiki/3H_Thymidine_Incorporation_Assay_for_Rat-1a_Cells

http://www.gladstone.ucsf.edu/gladstone/files/conklin/thymidine.html