Immunocytochemical analysis of mitogen responses of carp (Cyprinus carpio L.) peripheral blood leucocytes.

Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) responses of surface immunoglobulin-positive (sIg+) and surface immunoglobulin-negative (sIg-) carp peripheral blood leucocytes (PBL) were studied. sIg+ cell-enriched and depleted carp PBL populations (sIg+ and sIg- cell fractions, respectively) were obtained by magnetic cell sorting (MACS) and mitogenic stimulation in vitro was measured by 3H-thymidine incorporation. The mitogen responses of sIg+ and sIg- cells in non-separated carp PBL cultures were analysed by simultaneous detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) and sIg with the fluorescence microscope and flow cytometer. Flow cytometric determination of the percentage of sIg+ cells in combination with absolute cell counting, revealed an increase of sIg+ cells but not of sIg- cells after LPS stimulation while the number of sIg- cells and not of sIg+ cells was enhanced after PHA stimulation. LPS stimulation showed an increased 3H-thymidine incorporation in the sIg- cell fraction compared with non-separated cells and BrdU incorporation was observed in sIg- cells from LPS-stimulated cultures by fluorescence microscopy. However, flow cytometric analysis showed that mainly dull sIg+ cells and not sIg- cells are stimulated by LPS. These dull sIg+ cells were not sorted from sIg- cells with MACS and could apparently not be distinguished from sIg- cells by light microscopy. PHA stimulates sIg- cells and not sIg+ cells as was estimated by all techniques used.