A rapid and simple assay to determine the proliferation of larval and juvehile fish splenocytes

A simple assay to determine the proliferation of larval and juvehile fish s plenocytes using Alamar Blue (AB) was studied. Splenocytes were separated b y Percoll gradient from Japanese flounder Paralichthys olivaceus (1-year-ol d), then the proliferation of splenocytes stimulated by mitogen (concanaval in A: ConA, pokeweed mitogen: PWM, or lipopolysaccharide: LPS) was detected using AB. The relationship between number of splenocytes and specific abso rbance exhibited a positive significant correlation. The optimum condition of this assay was 5 x 10(5)cells/well of splenocytes with mitogens (ConA 10 0 mug/mL, PWM 10 mug/mL or LPS 1 mug/mL) for 72 h. Proliferation of each sp lenocyte from juveniles of Japanese flounder and larvae of Japanese parrotf ish Ophegnathus fasciatus and tiger puffer Takifugu rubripes was detectable by this assay.