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

Korinn E. Saker, Joan Kalnitsky, Robert M. Gogal, Daniel Ward

Research output: Contribution to journalReview article

9 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)567-571
Number of pages5
JournalAmerican journal of veterinary research
Volume62
Issue number4
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

Fingerprint

lymphocyte proliferation
Concanavalin A
Felidae
Cats
Lymphocytes
cats
concanavalin A
assays
Thymidine
Immunocompetence
thymidine
Poisons
lymphocytes
methodology
Cultured Cells
Flow Cytometry
Monoclonal Antibodies
immunocompetence
T-Lymphocytes
blood

All Science Journal Classification (ASJC) codes

  • veterinary(all)

Cite this

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title = "Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats",
abstract = "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.",
author = "Saker, {Korinn E.} and Joan Kalnitsky and Gogal, {Robert M.} and Daniel Ward",
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Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats. / Saker, Korinn E.; Kalnitsky, Joan; Gogal, Robert M.; Ward, Daniel.

In: American journal of veterinary research, Vol. 62, No. 4, 01.01.2001, p. 567-571.

Research output: Contribution to journalReview article

TY - JOUR

T1 - Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats

AU - Saker, Korinn E.

AU - Kalnitsky, Joan

AU - Gogal, Robert M.

AU - Ward, Daniel

PY - 2001/1/1

Y1 - 2001/1/1

N2 - 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.

AB - 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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