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

Phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) responses of su rface immunoglobulin-positive (sIg(+)) and surface immunoglobulin-nega tive (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-thymidin e incorporation. The mitogen responses of sIg(+) and sIg(-) cells in n on-separated carp PBL cultures were analysed by simultaneous detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) and sig with the fluor escence microscope and flow cytometer. Flow cytometric determination o f the percentage of sIg(+) cells in combination with absolute cell cou nting, revealed an increase of sIg(+) cells but not of sIg(-) cells af ter LPS stimulation while the number of sIg(-) cells and not of sIg(+) cells was enhanced after PHA stimulation. LPS stimulation showed an i ncreased H-3-thymidine incorporation in the sIg(-) cell fraction compa red with nonseparated cells and BrdU incorporation was observed in sIg (-) cells from LPS-stimulated cultures by fluorescence microscopy. How ever, flow cytometric analysis showed that mainly dull sIg(+) cells an d not sIg(-) cells are stimulated by LPS. These dull sIg(+) cells were not sorted from sIg(-) cells with MACS and could apparently not be di stinguished from sIg(-) cells by light microscopy. PHA stimulates sIg( -) cells and not sIg(+) cells as was estimated by all techniques used.