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Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats

Korinn E. SakerDepartment of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Joan KalnitskyOffice of Research and Graduate Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Robert M. Gogal JrOffice of Research and Graduate Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Daniel L. WardOffice of Research and Graduate Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061.

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Abstract

Objective—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 proliferative 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 felinespecific 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.

Conclusion 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. (Am J Vet Res 2001; 62:567–571)

Abstract

Objective—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 proliferative 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 felinespecific 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.

Conclusion 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. (Am J Vet Res 2001; 62:567–571)