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


METHODS
Blood samples from 10 clinically normal domestic shorthair cats.


METHODS
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 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml.


CONCLUSIONS
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.
Saker
K E
KE
Department of Large Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg 24061, USA.
Kalnitsky
J
J
Gogal
R M
RM
Ward
D L
DL
D000818:Animals; D016516:CD4-CD8 Ratio / Q000662:veterinary; D002415:Cats / Q000276:immunology*; D002470:Cell Survival / Q000187:drug effects; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D003124:Colorimetry / Q000379:methods / Q000662:veterinary*; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000276:immunology*; D016131:Lymphocyte Subsets / Q000737:chemistry; D008214:Lymphocytes / Q000276:immunology*; D008297:Male; D009000:Monocytes / Q000502:physiology; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D004247:DNA; D013936:Thymidine