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

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trook
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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


Last edited Feb 08, 2009, 22:58 PM by frasermoss
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