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 µL/well. The cells are maintained at 37ƒ C
(5% CO2) for approximately 24 - 48 hours. After this time, 10 µL 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 37ƒ 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. |
