High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.
High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: a potential source of error in analysis of blood samples utilizing fluorescein-conjugated reagents in flow cytometry.
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DOI：
10.1002/(sici)1097-0320(19990501)36:1
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发表时间：
1999-05
期刊：
影响因子：
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通讯作者：
E. Bedner;H. Halicka;Wei Cheng;T. Salomon;A. Deptała;W. Gorczyca;M. Melamed;Z. Darżynkiewicz
中科院分区：
文献类型：
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作者：
E. Bedner;H. Halicka;Wei Cheng;T. Salomon;A. Deptała;W. Gorczyca;M. Melamed;Z. Darżynkiewicz
背景：在使用基于DNA链断裂标记的凋亡检测商用试剂盒处理的外周血细胞样本中，通过流式细胞仪一致地观察到以高绿色荧光为特征的细胞亚群，其强度与凋亡细胞相似，但更均匀。标记的细胞没有其他的凋亡特征。无论使用何种固定剂，均可观察到标记，且在缺乏末端脱氧核苷酸转移酶的对照样本中明显。产生这种标记模式的所有试剂盒的共同之处是荧光素(F)偶联试剂、f-dUTP、f-亲和素或f-抗体的存在。方法采用激光扫描细胞术对标记细胞进行鉴定，并对标记机制进行研究。由于怀疑可能污染试剂的微量未结合f-异硫氰酸酯(FITC)是导致标记的原因，因此研究了FITC与白细胞的结合亲和力。用凝胶电泳法检测试剂中非偶联FITC的存在。结果经Giemsa染色后，强荧光物质为嗜酸性粒细胞，形态正常，无细胞凋亡迹象。荧光仅定位于细胞质颗粒内。在2 nM的FITC浓度下观察到嗜酸性粒细胞的标记，这比标记中性粒细胞、单核细胞或淋巴细胞所需的浓度低三个数量级以上。F-偶联试剂的凝胶电泳法显示FITC仅有轻微污染。结论(1)微量未结合的FITC污染试剂足以强烈标记嗜酸性粒细胞，从而在细胞凋亡分析和其他利用f标记抗体对血细胞的研究中引入实验偏差，例如在检测细胞因子方面。(2)浓度为2-500 nM的FITC可作为嗜酸性粒细胞的标志物；(3)由于对FITC的高亲和力，嗜酸性粒细胞(或来自这些细胞的蛋白质)可以作为一种手段，在试剂的制造过程中或使用前去除微量的未结合的FITC。
BACKGROUND In samples of peripheral blood cells processed using the commercial kits for detection of apoptosis based on DNA strand break labeling, a subpopulation of cells characterized by high green fluorescence, similar in intensity to that of apoptotic cells but more uniform, was consistently observed by flow cytometry. The labeled cells had no other features of apoptosis. The labeling was observed regardless of the fixative used and was evident in control samples lacking terminal deoxynucleotidyltransferase. Common to all the kits that generated this labeling pattern was the presence of fluorescein (f) conjugated reagents, f-dUTP, f-avidin, or f-antibody. METHODS Laser scanning cytometry was used to identify the labeled cells and study the mechanism of labeling. Because it was suspected that the traces of unconjugated f-isothiocyanate (FITC) that may contaminate the reagents were responsible for the labeling, FITC binding affinity to white blood cells was studied. Gel electrophoresis was used to detect the presence of unconjugated FITC in the reagents. RESULTS After staining with Giemsa, the strongly fluorescent objects were identified as eosinophils with normal morphology and no evidence of apoptosis. The fluorescence was localized exclusively within the cytoplasmic granules. Labeling of eosinophils was observed at 2 nM concentration of FITC, which was over three orders of magnitude lower than that needed to label neutrophils, monocytes, or lymphocytes. Gel electrophoresis of the f-conjugated reagents revealed only minor contamination with FITC. CONCLUSIONS (1) Trace amounts of unconjugated FITC contaminating the reagents are adequate to strongly label eosinophils thereby introducing experimental bias in analysis of apoptosis and in other studies on blood cells utilizing f-labeled antibodies, e.g., in detecting cytokines. (2) FITC at concentration 2-500 nM can be used as a marker of eosinophiles; (3) Because of high affinity to FITC, eosinophiles (or the protein from these cells) may serve as a means of removing traces of unconjugated FITC from the reagents during their manufacture or prior to use.