Alzhanov Damir T, Suchland Robert J, Bakke Antony C, Stamm Walter E, Rockey Daniel D
Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 97331, USA.
J Microbiol Methods. 2007 Jan;68(1):201-8. doi: 10.1016/j.mimet.2006.07.012. Epub 2006 Sep 25.
This manuscript describes a new technique for the microbiological cloning of chlamydia-infected cells using a fluorescence activated cell sorter (FACS). The approach exploits chlamydial acquisition of the fluorescent, Golgi-specific, stain 6-((N-7-(-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosine (C6-NBD-cer). This fluorescent lipid is delivered from the Golgi apparatus to the chlamydial inclusion membrane and then to the developmental forms within the inclusion in living, infected cells. Labeling with C6-NBD-cer results in easily identifiable chlamydial inclusions that can then be analyzed and sorted by FACS. This technique was used successfully to sort individual chlamydia-infected cells into individual wells of a culture dish and, in this experimental system, resulted in the isolation of cloned chlamydial isolates. FACS-based sorting was used to isolate clonal populations of prototype strains from Chlamydia trachomatis, C. caviae and C. suis. Recent clinical isolates were also successfully cloned using FACS. The procedure is simple and rapid, with single cloning cycles being completed 24 h post-culture of a sample. It is anticipated that FACS-based sorting of live chlamydia-infected cells will be a significant technical tool for the isolation of clonal populations of any chlamydial strain.
本手稿描述了一种使用荧光激活细胞分选仪（FACS）对衣原体感染细胞进行微生物克隆的新技术。该方法利用了衣原体摄取荧光性的、高尔基体特异性的染料6-((N-7-(-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基)氨基)-己酰基)鞘氨醇（C6-NBD-神经酰胺）。这种荧光脂质从高尔基体传递到衣原体包涵体膜，然后传递到活的感染细胞中包涵体内的发育形式。用C6-NBD-神经酰胺标记会产生易于识别的衣原体包涵体，然后可以通过FACS进行分析和分选。该技术已成功用于将单个衣原体感染细胞分选到培养皿的单个孔中，在这个实验系统中，导致了克隆衣原体分离株的分离。基于FACS的分选用于从沙眼衣原体、豚鼠衣原体和猪衣原体中分离原型菌株的克隆群体。最近的临床分离株也使用FACS成功克隆。该程序简单快速，单个克隆周期在样品培养24小时后完成。预计基于FACS对活的衣原体感染细胞进行分选将成为分离任何衣原体菌株克隆群体的重要技术工具。
