Quantification of infiltrating leukocytes in the lateral line after diverse treatments: the chemically induced inflammation (ChIn) assay. (a) Schematic view of a 3 days postfertilization (dpf) larva. The boxed area corresponds to the horizontal myoseptum (line). An area of approximately 10 cell diameters is delimited around the myoseptum (dotted red lines) and corresponds to the area where leukocytes were counted in all manual quantification experiments. (b) Significant induction of leukocyte recruitment to the lateral line by copper treatment. The graph shows average leukocyte numbers in the lateral line in negative controls (untreated fish or cadmium chloride-treated fish) and in copper-treated fish. (c) The effect of copper on leukocyte recruitment to the lateral line is concentration dependent. (d) Effectiveness of other metals in the ChIn assay. (e) Neomycin, at concentrations that eliminate hair cells, also induces leukocyte recruitment, but less effectively than copper. (f) Larvae of different ages, from 56 to 128 hpf, were exposed to 10 μM CuSO4 and were monitored for leukocytes present at the myoseptum every 20 minutes thereafter until 120 minutes. Fish at all stages analyzed showed similar behaviors and exhibited increased presence of leukocytes at the lateral line by 20 minutes after initiating exposure to copper. For all experiments, at least 15 larvae were used for each condition. ***P < 0.001.