Verburg-Van Kemenade B M, Saeij J P, Flik G, Willems P H
Cell Biology and Immunology Laboratory, Institute of Animal Sciences, Agricultural University, Wageningen, The Netherlands.
J Exp Biol. 1998 Feb;201(Pt 4):591-8. doi: 10.1242/jeb.201.4.591.
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.
为了测量细胞反应以及细胞溶质Ca2+水平升高（[Ca2+]i）的影响，鲤鱼外周血白细胞（PBL）被装载了荧光细胞内Ca2+指示剂Fluo-3和Fura-2。使用流式细胞术测量淋巴细胞对T细胞有丝分裂原（植物血凝素，PHA）、对B细胞有丝分裂原（脂多糖，LPS）以及对与鲤鱼Ig单克隆抗体进行免疫球蛋白（Ig）交联的反应。PHA对T细胞的刺激以及膜Ig交联对B细胞的刺激均引起[Ca2+]i的快速升高。LPS对B细胞的刺激与[Ca2+]i的增加无关。根据反应细胞的百分比判断，得出结论：所有Ig阳性淋巴细胞通过升高[Ca2+]i对Ig交联产生反应。在单细胞水平，每6秒使用数字成像显微镜跟踪Fura-2装载细胞的反应。显示自发[Ca2+]i振荡的细胞和非振荡细胞对刺激均以[Ca2+]i增加做出反应，有时，在已经振荡的细胞中，伴随着振荡频率和/或振幅的增加。这些结果表明，PBL激活后的细胞内Ca2+反应与哺乳动物相似，是研究细胞特异性调节的有力工具。
