To measure cellular responses and the involvement of increased cytosolic Ca2+ levels ([Ca2+]i), peripheral blood leukocytes (PBL) of carp were loaded with the fluorescent intracellular Ca2+ indicators Fluo-3 and Fura-2. Responses of lymphocytes to T-cell mitogen (phytohaemagglutinin, PHA), to B-cell mitogen (lipopolysaccharide, LPS) and to immunoglobulin (Ig) cross-linking with a monoclonal antibody to carp Ig were measured using flow cytometry. Both T-cell stimulation by PHA and B-cell stimulation by membrane Ig cross-linking evoked a rapid elevation of [Ca2+]i. B-cell stimulation by LPS was not linked to an increase in [Ca2+]i. As judged by the percentage of reacting cells, it was concluded that all Ig-positive lymphocytes reacted to Ig cross-linking by elevating [Ca2+]i. At the single-cell level, the reactions of Fura-2-loaded cells were followed every 6 s using digital imaging microscopy. Both cells displaying spontaneous [Ca2+]i oscillations and non-oscillating cells responded to stimulation with an increase in [Ca2+]i, sometimes, in already oscillating cells, accompanied by an increase in frequency and/or amplitude of the oscillations. These results show that intracellular Ca2+ responses of PBL upon activation resemble those in mammals and form a powerful tool for studies into cell-specific regulation.

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