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
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;