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Analysis of avian lymphocyte proliferation by a new, simple, nonradioactive assay (lympho-pro)
R M Gogal1, S A Ahmed, C T Larsen
1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg 24061, USA.
Insights
A new, non-radioactive assay effectively measures T-cell proliferation in chickens. This method simplifies immune function assessment in avian research and diagnostics, offering a faster, safer alternative to traditional techniques.
Area of Science:
- * Avian immunology
- * Cellular immunology
- * Veterinary diagnostics
Background:
- * Assessing T-cell-mediated immune functions in chickens is challenging due to difficulties in isolating pure lymphocyte populations and the use of radioactive isotopes.
- * Current methods for determining lymphocyte proliferation in avian species are not routinely performed, hindering research and diagnostics.
Purpose of the Study:
- * To optimize a method for isolating and enriching avian lymphocyte populations from chicken peripheral blood.
- * To develop and validate a nonradioactive lymphocyte proliferation assay for avian T-cell immune function assessment.
Main Methods:
- * Lymphocyte isolation using a combination of slow-speed centrifugation and a "swirl" collection technique.
- * Adaptation and comparison of a colorimetric/fluorometric assay (lympho-pro) with the traditional [3H]thymidine incorporation assay.
- * Stimulation of lymphocytes with concanavalin A (Con A) or phorbol 12-myristate 13-acetate + ionomycin in chickens of different ages and genetic strains.
Main Results:
- * The lympho-pro assay demonstrated a similar pattern of lymphocyte proliferation compared to the [3H]thymidine assay.
- * Younger chickens exhibited higher levels of mitogen-induced lymphocyte proliferation than adult chickens of the same genetic strain.
- * The lympho-pro assay allows for subsequent characterization of proliferated cells via flow cytometry due to its non-lytic nature.
Conclusions:
- * The developed nonradioactive lympho-pro assay is a viable and advantageous alternative for assessing T-cell-mediated immunity in avian research and diagnostics.
- * This assay offers advantages of rapidity, non-toxicity, and compatibility with downstream cellular analysis, simplifying immune function assessments in chickens.
Abstract:
An assessment of T-cell-mediated immune functions (i.e., lymphocyte proliferation assay) in the chicken, unlike the determination of antibody levels, is not routinely performed. This is primarily because of difficulties in the isolation of relatively pure populations of lymphocytes and the use of radioactive isotopes. To address these issues, the goals of our study were to optimize a method for isolating and enriching avian lymphocyte populations and to develop a nonradioactive lymphocyte proliferation assay. To accomplish these goals, we used a multiple slow-speed centrifugation technique combined with a "swirl" collection technique for lymphocyte isolation from chicken peripheral blood. After a fraction enriched with lymphocytes was obtained, a simple, rapid colorimetric and fluorometric assay (lympho-pro) to indirectly determine mitogen-induced proliferation was adapted and compared with the "Gold Standard" [3H]thymidine. Chickens of different ages and two genetic strains were used in this study. Lymphocytes were stimulated with various concentrations of concanavalin A (Con A, T-cell mitogen) or phorbol 12-myristate 13-acetate + ionomycin (pan lymphocyte mitogen). Our studies showed that the pattern of lymphocyte proliferation assessed by the Alamar blue-based lympho-pro assay was similar to the [3H]thymidine incorporation assay. Younger birds had higher levels of mitogen-induced proliferation when compared with adults of the same genetic strain. Because the lympho-pro assay, unlike [3H]thymidine, does not require lysis of cells to assess proliferation, cells that have undergone stimulation/proliferation can be subsequently characterized by staining with antibodies against cell surface antigens and analysis by flow cytometry. Another notable advantage of the lympho-pro assay is the rapidity of assessment and nontoxicity. In conclusion, this assay may be of value in assessing some aspects of T-cell-mediated immunity in both avian research and avian medicine diagnostic settings.
