Koumans-van Diepen J C, Harmsen E G, Rombout J H
Department of Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, Netherlands.
Vet Immunol Immunopathol. 1994 Aug;42(2):209-19. doi: 10.1016/0165-2427(94)90009-4.
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.
研究了鲤鱼外周血白细胞（PBL）表面免疫球蛋白阳性（sIg+）和表面免疫球蛋白阴性（sIg-）细胞对植物血凝素（PHA）和脂多糖（LPS）的反应。通过磁珠细胞分选（MACS）获得富含sIg+细胞和去除sIg+细胞的鲤鱼PBL群体（分别为sIg+和sIg-细胞组分），并通过3H-胸腺嘧啶核苷掺入法测定体外促有丝分裂刺激。通过荧光显微镜和流式细胞仪同时检测掺入的5-溴-2'-脱氧尿苷（BrdU）和sIg，分析未分离的鲤鱼PBL培养物中sIg+和sIg-细胞的促有丝分裂原反应。结合绝对细胞计数，通过流式细胞术测定sIg+细胞的百分比，结果显示LPS刺激后sIg+细胞增加，而sIg-细胞未增加，而PHA刺激后sIg-细胞数量增加，sIg+细胞未增加。与未分离的细胞相比，LPS刺激显示sIg-细胞组分中3H-胸腺嘧啶核苷掺入增加，通过荧光显微镜观察到LPS刺激培养物中的sIg-细胞中有BrdU掺入。然而，流式细胞术分析表明，LPS主要刺激暗淡的sIg+细胞，而不是sIg-细胞。这些暗淡的sIg+细胞未通过MACS从sIg-细胞中分离出来，并且通过光学显微镜显然无法与sIg-细胞区分开来。所有使用的技术均估计PHA刺激sIg-细胞，而不是sIg+细胞。
