Quantification of mitogen induced human lymphocyte proliferation: comparison of alamarBlue assay to 3H-thymidine incorporation assay.
Proliferation of human lymphocytes in response to various stimuli has traditionally been assessed by measuring uptake of radiolabeled nucleotides such as 3H-thymidine. We have evaluated a fluorometric assay, which uses the commercially available reagent, alamarBlue, as a potential substitute for the 3H-thymidine assay in measuring proliferation of human lymphocytes. In this assay, alamarBlue is added to a population of cells where it is reduced by mitochondrial enzyme activity. The reduced form of the reagent is fluorescent and can be quantitatively detected. The safety and convenience of the alamarBlue assay make it very attractive for use in the clinical laboratory. In this study peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated using the mitogen Concanavalin A, and proliferation was assessed using either the 3H-thymidine or the alamarBlue assay. The alamarBlue assay reliably detects human PBMC and we found that the linear range of detection was 10(4) cells/well (96-well plate) to 5 x 10(5) cells/well. Detection of human PBMC is highly reproducible and the alamarBlue assay may be suitable in a number of applications where detection or relative quantitation of human PBMC is required. The alamarBlue assay also detected mitogen induced proliferation of PBMC although with a significantly lower level of sensitivity than the standard 3H-thymidine assay.
de Fries
R
R
Medical Sciences Division, Hitachi Chemical Research Center, Inc., Irvine, California 92715, USA.
Mitsuhashi
M
M
D002452:Cell Count / Q000379:methods; D004396:Coloring Agents; D003208:Concanavalin A; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008213:Lymphocyte Activation; D008722:Methods; D010078:Oxazines; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013936:Thymidine / Q000378:metabolism*; D014316:Tritium; D014966:Xanthenes
D004396:Coloring Agents; D005456:Fluorescent Dyes; D010078:Oxazines; D011933:Reagent Kits, Diagnostic; D014966:Xanthenes; D014316:Tritium; D003208:Concanavalin A; C005843:resazurin; D013936:Thymidine