A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages
A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages
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DOI：
10.1002/cyto.a.22706
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发表时间：
2015-09-01
期刊：
影响因子：
3.7
通讯作者：
Rosania, Gus R.
中科院分区：
文献类型：
--
作者：
Keswani, Rahul K.;Yoon, Gi S.;Rosania, Gus R.
氯法齐明（CFZ）是一种光学活性的红色化疗剂，FDA批准用于治疗麻风病，并被列入世界卫生组织的基本药物清单。有趣的是，CFZ在巨噬细胞中大量积累，在动物和人类口服药物后，CFZ在巨噬细胞中形成晶体样药物包涵体（CLDIs）。对由驻留组织巨噬细胞形成的CLDIs的荧光光谱的分析表明，当CFZ作为CLDIs积累时，相对于CFZ的可溶性和游离碱晶体形式，CFZ经历荧光激发（从Ex：540-570至560-600 nm）和发射（Em：560-580至640-700 nm）信号的红移。使用落射荧光显微术，基于在640 nm激发和670 nm发射处的平均荧光信号的>3倍增量，相对于CLDI ㈠细胞，可以鉴定CLDI ⑴细胞。类似地，CLDI（+）细胞可以通过流式细胞术鉴定，基于使用640 nm激发激光和>600 nm发射检测器的平均荧光信号的>100倍增量。CLDI的荧光激发和发射与细胞活力染料如碘化丙啶和4，6-二脒基-2-苯基吲哚二盐酸盐（DAPI）、细胞染色染料如Hoechst 33342（细胞核）和FM 1-43（质膜）以及用于免疫表型分析的许多其他荧光标记抗体的荧光激发和发射正交。在体内，腹膜渗出液中>85%的CLDI（+）细胞是F4/80（+）巨噬细胞，肺泡渗出液中>97%的CLDI（+）细胞是CD 11 c（+）。最重要的是，细胞的活力受CLDIs存在的影响最小。因此，这些结果确立了CLDIs中的CFZ荧光适用于定量流式细胞术表型分析和异生物素螯合巨噬细胞的功能研究。(c)2015年国际细胞计数促进学会
Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages. (c) 2015 International Society for Advancement of Cytometry