Denholm E M, Stankus G P
Department of Pharmacology, Sterling Winthrop Pharmaceutical Research Division, Collegeville, Pennsylvania, USA.
Cytometry. 1995 Apr 1;19(4):366-9. doi: 10.1002/cyto.990190412.
Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein-acetoxymethyl ester (calcein-AM) or 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), then assayed for their ability to migrate to zymosan-activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF-AM decreased the number of cells responding to ZAS, while calcein-AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF-AM on cell migration increased with probe concentration (0.1-1.0 microM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF-AM and those labeled with calcein-AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods.
荧光探针已被用于标记白细胞，以进行细胞迁移的体内和体外研究；然而，此类探针对迁移的影响尚未确定。本研究的目的是检测两种常用荧光探针对白细胞趋化性的影响。用乙酰氧基甲基钙黄绿素（calcein-AM）或2',7'-双（2-羧乙基）-5-（和6）-羧基荧光素乙酰甲酯（BCECF-AM）标记J774巨噬细胞，然后检测它们向酵母聚糖激活血清（ZAS）迁移的能力。通过两种方法对细胞迁移进行定量：细胞的视觉计数和测量细胞荧光。使用细胞计数，未标记和荧光标记巨噬细胞的比较表明，BCECF-AM减少了对ZAS作出反应的细胞数量，而calcein-AM基本没有影响。两种探针均未显著影响仅迁移到培养基中的细胞数量。BCECF-AM对细胞迁移的抑制作用随探针浓度（0.1-1.0 microM）和细胞荧光的增加而增强。两种探针均未影响细胞活力。与视觉计数获得的结果相反，测量迁移细胞的荧光未显示用BCECF-AM标记的巨噬细胞和用calcein-AM标记的巨噬细胞的趋化反应之间存在显著差异。这些实验表明，荧光探针可影响趋化反应，并且当仅通过自动化方法对趋化性进行定量时，可能检测不到这些探针的抑制活性。
