Batisse Chantal, Marquet Jeanine, Greffard Anne, Fleury-Feith Jocelyne, Jaurand Marie-Claude, Pilatte Yannick
EMI 99-09 INSERM, Faculté de Médecine Paris XII, Créteil, France.
Cytometry A. 2004 Dec;62(2):81-8. doi: 10.1002/cyto.a.20094.
Changes in cell surface glycosylation that accompany apoptosis are thought to be involved in the recognition and removal of apoptotic cells by phagocytes, but in most instances these changes are ill defined. To improve our understanding of this phenomenon, we designed a trivariate flow cytometry procedure that allows direct comparison of cell surface glycosylation in apoptotic and viable cells.
The annexin V/propidium iodide assay has been adapted for cell surface glycosylation analysis by combining the use of these two reagents with biotinylated lectins, and this has been used to investigate camptothecin-induced apoptosis in U-937 cells.
Although numerous lectins are potent inducers of apoptosis, we found that it is possible to determine lectin concentrations that produce interpretable data without inducing significant cytotoxicity even when using apoptogenic lectins. That apoptosis is associated with a marked decrease in cell surface sialylation was confirmed by using the sialic acid-specific lectins Maackia amurensis agglutinin and Sambucus nigra agglutinin. These observations were corroborated by lectin blotting analysis with the same lectins.
Species- and cell-dependent altered glycosylation patterns are likely to be associated with different modes of apoptosis. The easy and versatile method described in this report should be useful for exploring this field.
凋亡过程中伴随的细胞表面糖基化变化被认为参与了吞噬细胞对凋亡细胞的识别和清除，但在大多数情况下，这些变化并不明确。为了更好地理解这一现象，我们设计了一种三变量流式细胞术程序，可直接比较凋亡细胞和活细胞的细胞表面糖基化情况。
通过将膜联蛋白V/碘化丙啶检测法与生物素化凝集素结合，对其进行了改进以用于细胞表面糖基化分析，并已用于研究喜树碱诱导的U-937细胞凋亡。
尽管许多凝集素是凋亡的有效诱导剂，但我们发现即使使用促凋亡凝集素，也有可能确定能产生可解释数据而不诱导明显细胞毒性的凝集素浓度。使用唾液酸特异性凝集素黑果腺肋花楸凝集素和接骨木凝集素证实了凋亡与细胞表面唾液酸化的显著减少有关。用相同的凝集素进行凝集素印迹分析也证实了这些观察结果。
物种和细胞依赖性的糖基化模式改变可能与不同的凋亡模式有关。本报告中描述的这种简单通用的方法应有助于探索该领域。
