Czepluch Frauke S, Olieslagers Servé J F, Waltenberger Johannes
Department of Cardiology, Cardiovascular Research Institute Maastricht, University of Maastricht, P. Debyelaan 25, P.O. Box 5800, 6202 AZ Maastricht, Maastricht, The Netherlands.
Biochem Biophys Res Commun. 2007 Sep 21;361(2):410-3. doi: 10.1016/j.bbrc.2007.07.018. Epub 2007 Jul 16.
For rapid chemotaxis quantification, cell prelabelling is often performed with the fluorochrome calcein acetomethylester (calcein AM). We investigated whether calcein AM-prelabelling is reliable for monocyte migration analysis. Human monocytes were either preexposed to calcein AM or unlabelled. Monocyte migration towards the potent chemoattractants transforming growth factor-beta1 (TGF-beta1) and N-formyl-Methionin-Leucin-Phenylalanin (fMLP) was assessed using a 48-well micro-chemotaxis chamber. For quantification, cells were visualized by light microscopy and counted. Surprisingly, random migration of calcein AM-prelabelled cells was significantly impaired compared to the unlabelled control. Accordingly, monocyte chemotaxis towards either TGF-beta1 or fMLP dramatically declined. Adherence of calcein AM-labelled monocytes on plastic was also significantly decreased compared to control cells. As adhesion is regarded as an essential component of monocyte migration, the reduced migration observed in calcein AM-labelled monocytes might be explained by a fluorochrome-induced adhesion defect. Therefore, use of the fluorochrome calcein AM cannot be recommended for functional testing of monocytes.
为了快速进行趋化性定量分析，细胞预标记通常使用荧光染料钙黄绿素乙酰甲酯（calcein AM）。我们研究了calcein AM预标记对于单核细胞迁移分析是否可靠。将人单核细胞要么预先暴露于calcein AM，要么不进行标记。使用48孔微量趋化性小室评估单核细胞向强效趋化因子转化生长因子-β1（TGF-β1）和N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）的迁移。为了进行定量分析，通过光学显微镜观察细胞并计数。令人惊讶的是，与未标记的对照相比，calcein AM预标记细胞的随机迁移明显受损。相应地，单核细胞对TGF-β1或fMLP的趋化性显著下降。与对照细胞相比，calcein AM标记的单核细胞在塑料上的黏附也显著降低。由于黏附被认为是单核细胞迁移的一个重要组成部分，在calcein AM标记的单核细胞中观察到的迁移减少可能是由荧光染料诱导的黏附缺陷所解释。因此，不推荐使用荧光染料calcein AM进行单核细胞的功能测试。
