Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes
Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes
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DOI：
10.1016/s0022-1759(00)00189-7
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发表时间：
2000-05-26
影响因子：
2.2
通讯作者：
Nash, GB
中科院分区：
文献类型：
--
作者：
Abbitt, KB;Rainger, GE;Nash, GB
荧光染料有助于白细胞的可视化，可用于黏附的活体研究或利用全血的体外研究。我们使用了基于体外流动的检测方法来研究三种荧光染料（吖啶橙，AO，5 - 100微克/毫升；钙黄绿素 - AM，C - AM，5 - 20微克/毫升；罗丹明6G，R6G，10 - 100微克/毫升）对分离的中性粒细胞和单核细胞的黏附和迁移的影响。AO对在血小板包被表面呈现的P - 选择素上滚动的中性粒细胞的数量或速度影响很小。然而，AO确实导致了滚动向固定的剂量和时间依赖性转变。用抗CD18抗体预处理中性粒细胞可阻止这种向固定黏附的转变，表明β(2)整合素被AO激活。C - AM对中性粒细胞的行为影响很小，但在最高浓度时往往会导致一些固定。R6G不影响与血小板单层结合的中性粒细胞的数量或滚动百分比，但中性粒细胞的滚动速度以剂量依赖性方式增加。这些染料都没有损害中性粒细胞通过从滚动转变为固定黏附来对甲酰肽作出反应的能力。C - AM和R - 6G都没有减少与肿瘤坏死因子刺激的内皮细胞结合的流动中性粒细胞或单核细胞的数量。有趣的是，R - 6G抑制单核细胞但不抑制中性粒细胞的跨内皮迁移，而C - AM不影响这两种细胞类型的迁移。在设计任何实验方案时都应考虑染料的剂量依赖性效应。AO似乎不是一种适合用于黏附研究的染料。R6G和C - AM可以在约10微克/毫升的浓度下使用（在此浓度下细胞可以清晰可视化），尽管R6G特异性地抑制单核细胞的迁移反应。（C）2000年爱思唯尔科学出版社。保留所有权利。
Fluorescent dyes assist visualisation of leukocytes for intravital studies of adhesion or for in vitro studies utilising whole blood. We have used in vitro flow-based assays to investigate the effects of three fluorescent dyes (acridine orange, AO, 5-100 mu g/ml; calcein-AM, C-AM, 5-20 mu g/ml; rhodamine 6G, R6G, 10-100 mu g/ml) on adhesion and migration of isolated neutrophils and mononuclear cells. AO had little effect on the number or velocity of neutrophils rolling on P-selectin presented by a surface coated with platelets. However, AO did cause a dose- and time-dependent conversion of rolling to immobilisation. Pretreatment of neutrophils with an antibody against CD18 prevented this conversion to stationary adhesion, indicating that beta(2) integrins were activated by AO. C-AM had little effect on neutrophil behaviour, but tended to cause some immobilisation at the highest concentration. R6G did not affect the number of neutrophils that bound to the platelet monolayer or the percentage rolling, but the rolling velocity of the neutrophils was increased in a dose-dependent manner. None of the dyes impaired the ability of neutrophils to respond to formyl peptide by converting from rolling to stationary adhesion. Neither C-AM nor R-6G reduced the number of flowing neutrophils or mononuclear cells binding to endothelial cells stimulated with tumour necrosis factor. Interestingly, R-6G inhibited transendothelial migration of mononuclear cells but not neutrophils, while C-AM did not affect transmigration of either cell type. The dose-dependent effects of dyes should be taken into consideration when designing any experimental protocol. AO does not appear to be a suitable dye for adhesion studies. R6G and C-AM can be used at similar to 10 mu g/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells. (C) 2000 Elsevier Science B.V. All rights reserved.