Objectives - To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferate activity of feline lymphocytes. Sample Population - Blood samples from 10 clinically normal domestic shorthair cats. Procedure - Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry. Results - Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 μg of Con-A/ml were submitogenic, and 100 μg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 μg of Con-A/ml. Conclusions and Clinical Relevance - These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.
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