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Published on: October 13, 2010
Use of a simple light absorbance assay to measure lymphocyte proliferation
Z H Gao1, W A Briggs, N R Rose
1Johns Hopkins Hospital, Department of Surgery, Baltimore, Maryland 21205, USA.
Insights
A new light absorbance assay effectively measures human lymphocyte proliferation and viability. This method offers a simpler, faster, and safer alternative to traditional tritiated thymidine incorporation assays.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Lymphocyte proliferation is crucial for adaptive immunity.
- Tritiated thymidine incorporation is the standard method for assessing lymphocyte proliferation.
- This method is costly, time-consuming, and involves hazardous materials.
Purpose of the Study:
- To evaluate a novel light absorbance assay for measuring lymphocyte proliferation and viability.
- To compare the light absorbance assay with established methods like tritiated thymidine incorporation, MTS/PMS assay, and trypan blue exclusion.
Main Methods:
- Human lymphocytes were cultured with stimuli.
- Light absorbance was measured using an ELISA reader at 450 nm/air-550 nm/air.
- Proliferation and viability were assessed using tritiated thymidine incorporation, MTS/PMS assay, and trypan blue exclusion.
Main Results:
- The light absorbance assay showed good correlation with cell proliferation between 48-120 hours of culture.
- The assay also correlated well with cell viability after 72 hours.
- Results were validated against MTS/PMS and trypan blue exclusion, confirming the assay detects proliferation, not just dead cells.
- The light absorbance assay demonstrated sensitivity comparable to tritiated thymidine incorporation for low proliferation levels.
Conclusions:
- The light absorbance assay provides a sensitive, reliable, and cost-effective method for assessing lymphocyte proliferation and viability.
- This assay offers a practical alternative to traditional methods, reducing costs, time, and safety hazards.
- The simplicity and efficiency of the light absorbance assay make it suitable for various research and diagnostic applications.
Abstract:
The proliferative response of human lymphocytes to stimuli such as foreign histocompatibility antigens or mitogens is generally assessed by measuring the amount of tritiated thymidine which the cells incorporated in culture. In this paper, the possibility of assessing lymphocyte proliferation and viability by an empirical assay, using measurement of light absorbance on a ELISA reader in the yellow wave length (450 nm/air-550 nm/air), has been studied. The correlation of these measurements with a colormetric viability assay using MTS/PMS, with tritiated thymidine incorporation and with trypan blue exclusion viability counting, was determined. The results showed that the light absorbance assay correlated well with cell proliferation during 48-120 hours culture period and with cell viability after a 72 hour period. The MTS/PMS colormetric assay as well as trypan blue exclusion cell counting confirmed that the light absorbance assay was not merely caused by dead cells. This data confirm that the light absorbance assay is sufficiently sensitive to low levels of proliferation to allow detection of such responses at least as effectively as thymidine incorporation. The light absorbance assay procedure avoids the expense, time and hazards associated with scintillation counting, and is simple to perform without the necessity for reagents and preparative steps required by other assays.
