Abstract: In this study，sterlet（Acipenser ruthenus）was chosen as the model experimental material，different concentrations of lymphocyte separation solution were used to isolate the peripheral blood lymphocytes of sturgeon and study their optimal proliferate response condition. Phytohemagglutinin（PHA），concanavalin A（ConA）and lipopolysaccharide（LPS）were utilized as lymphocyte proliferation mitogen respectively. According to five factors and five levels orthogonal experimental design L25（56），the conditions of sturgeons peripheral blood lymphocytes proliferate response were optimized using Enhanced Cell Counting Kit-8（enhanced CCK-8 or WST-8）. Five factors were selected to explore the optimal response condition，including culture time，culture temperature，cell concentration，fetal bovine serum（FBS）concentration，and mitogen concentration. The results showed that 70% Percoll（1.092 g/mL）used as the sturgeon lymphocyte separation solution had the best separation effect. The optimal conditions for proliferation were 3.625×106 initial cell concentration，20 μg/mL PHA or 50 μg/mL ConA or 10 μg/mL LPS，10% - 20% FBS，20-25℃，and culturing for 2 days.

Key words: sturgeons, peripheral blood lymphocytes, lymphocyte isolation, proliferation, PHA, ConA, LPS, CCK-8