IMMUNOCYTOCHEMICAL ANALYSIS OF MITOGEN RESPONSES OF CARP (CYPRINUS-CARPIO L) PERIPHERAL-BLOOD LEUKOCYTES

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 H-3-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 H-3-thymidine incorporation in the sIg(-) cell fraction compared with nonseparated 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.