氯法齐明(CFZ)是一种光学活性,FDA批准用于治疗麻风病的红色化学治疗剂,并在世界卫生组织的基本药物清单上。有趣的是,CFZ在巨噬细胞中大量积累,在动物和人类中口服施用药物后,在巨噬细胞中形成晶体样药物内含物(CLDI)。对由常驻组织巨噬细胞形成的CLDIs的荧光光谱的分析表明,CFZ,当作为CLDI累积时,相对于CFZ的可溶性和游离碱晶体形式,经历荧光激发(从Ex:540-570至560-600nm)和发射(Em:560-580至640-700nm)信号的红移。使用落射荧光显微镜,可以识别CLDI(+)细胞,相对于CLDI(-)单元格,基于在激发640nm处和在670nm处发射的平均荧光信号的>3倍增量。同样,CLDI(+)细胞可以通过流式细胞术鉴定,基于使用640nm的激发激光和>600nm的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碘化丙啶和4,6-二氨基-2-苯基吲哚二盐酸盐(DAPI)等细胞活力染料的荧光激发和发射正交,细胞染色染料如Hoechst33342(细胞核)和FM1-43(质膜),以及许多其他用于免疫表型分析的荧光标记抗体。在体内,腹膜渗出物中>85%的CLDI()细胞是F4/80()巨噬细胞,肺泡渗出物中>97%的CLDI()细胞是CD11c()。最重要的是,CLDIs对细胞活力的影响最小。因此,这些结果证明CLDIs中的CFZ荧光适用于定量流式细胞仪表型分析和异种生物隔离巨噬细胞的功能研究。
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.