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NON-RADIOACTIVE SYSTEM FOR MEASURING CELL PROLIFERATION
Summary:
Currently, a non-radioactive system for measuring cell proliferation is being established within one of the University of Michigan (U-M) biomedical research laboratories. The laboratory historically utilized tritiated thymidine incorporation as the methodology for documenting deoxyribonucleic acid DNA synthesis. The proposed assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases found in viable cells. The benefits include the simplicity and rapidity with which multiple samples can be assayed, and elimination of organic and radioactive waste. The major disadvantage at this time is the need for a microtiter counter.
Applicable Regulations
10 CFR 20 Subpart K.

Overview of Procedure
Historically, the laboratory used the incorporation of tritiated thymidine as an indirect measure of cell proliferation. The procedure was somewhat cumbersome and required the use of radioactive material. Researchers are attempting to utilize a new system based on a colorimetric assay for the quantification of cell proliferation and cell viability. This assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Radioactive material is not required and many samples may be assayed simultaneously.

Waste Minimization Procedure
Study cells are cultured in microtiter plates in a final volume of 100 L/well. The cells are maintained at 37 C (5% CO2) for approximately 24 - 48 hours. After this time, 10 L per well of the cell proliferation reagent WST-1 is added. Cells are incubated for 0.5 to 4 hours in the 5% CO2 chamber at 37 C. Absorbance of the samples is measured against the background control using a microtiter plate (Enzyme Linked Immunosorbent Assay-ELISA) reader. The wave length for measuring the absorbance of the formosan product is between 420-480 nm. The reference wavelength should be more than 600 nm.

When using tritiated thymidine the maximum number of samples that could be measured in the plates was 12, thus generating a significant quantity of waste from solutions and plastic ware. The new technique can measure 96 separate samples on one ELISA plate. In addition, the new method does not require radioactivity; thus the entire radioactive waste stream associated with thymidine is eliminated. No volatile organic solvents are used for solubilization in the new method.

Known Limitations
Initial experiments have indicated that the sensitivity may not be as high as with thymidine, but may be attributed to the researchers inexperience with the new method. Researchers are currently working through the assay to enhance its sensitivity.

Safety & Health Precautions/Personal Protective Equipment
Follow all applicable safety and health protocols and regulations as established by your institution.

Benefits

  • Ability to measure multiple samples on one ELISA plate;
  • Elimination of radioactive material;
  • Elimination of organic solvents;
  • Increased sensitivity over the tetrazolium salt systems developed in the past;
  • Straightforward, simple and quick procedure;
  • One ELISA plate reader can support several laboratories.

Disadvantages
The main disadvantage noted by the researchers is the need to utilize an ELISA plate reader. The equipment is costly and may not be readily available in all laboratories. Therefore, additional time may need to be factored into the total research time necessary for locating and using an ELISA plate reader.

Project Related Costs
There is an initial cost of $5,000 for the ELISA plate reader. The WST-1 cell proliferation reagent costs are slightly higher compared to the same number of samples requiring tritiated thymidine reagent. Although reagent cost is slightly higher using the non-radioactive system, a total cost savings is still realized by the elimination of the need for scintillation vials and cocktails. A total of $2.92 per sample, at 50 samples per week, or $7,592 per year is saved. In addition, there are no disposal costs for radioactive materials and chemicals, which will add to the overall cost savings when using the non-radioactive system. The payback period will vary depending upon the number of laboratories using the plate reader. In addition, there are no disposal costs for radioactive materials and chemicals, which will add to the overall cost savings when using the non-radioactive system.

The non-radioactive system also requires fewer person-hours to perform, therefore resulting in a long-term cost savings of research time and money.

 

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