A rapid and simple assay to determine the blastogenesis of fish larval splenocytes using Alamar Blue

1 A rapid and simple colorimetric assay to determine the blastogenesis of fish larval splenocytesusing Alamar Blue was studied. Splenocyteswere separated by Percoll gradient from Japanese flounder(one year old), then the blastogenesis of splenocytesstimuiated by mitogen ( concanavalinA, pokeweed mitogen, or lipopolysaccharide) as detected using Alamar Blue. The relationship between number of splenocytesand specific absorbance at 72h incubation exhibited a positive, highly significant correlation of p< 0.001. Optimum condition of this assay at 5× 10 5 cells/well of separated splenocyteswith mitogen ( concanavalinA 100μg/ml, pokeweed mitogen10μg/ml, or lipopolysaccaride 1μg/ml) for 72h at 25℃. Splenocytesseparation procedure was not required for this blastogenesis assay. Blastogenesis of each splenocytefrom larva of Japanese flounder, Japanese parrotfish, and tiger puffer was detectable by this Alamar Blue assay. These results suggested that colorimetric assay using Alamar Blue might be of benefit in a host defense test of cultured fish larva against viral and/or bacterial infections.