Podgorny Oleg V, Polina Nadezhda F, Lazarev Vassili N
Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia.
Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia.
Methods Mol Biol. 2019;2042:137-150. doi: 10.1007/978-1-4939-9694-0_10.
Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the microscope. Individual C. trachomatis-infected cells are harvested into separate wells with a pregrown host cell monolayer. Inclusions in harvested cells maturate, and the released elementary bodies infect the host cell monolayer, thus initiating propagation of single inclusion-derived Chlamydia. The method can be used for generation of microbiological clones of C. trachomatis and recovery of transformants and mutants. Isolated single Chlamydia-infected cells can also be examined by diverse downstream molecular assays to reveal unknown features of the Chlamydia replication at a single inclusion level.
除了常规用于从固定组织切片中捕获纯细胞群体以进行各种下游分子分析外，激光显微切割还能够分离单个活细胞。在此，我们描述了一种使用激光显微切割系统分离单个沙眼衣原体感染细胞的方法，其中通过重力捕获切割后的样本。以低感染复数感染沙眼衣原体的细胞用荧光高尔基体特异性探针BODIPY FL C5-神经酰胺进行标记，以便于在显微镜下识别含有衣原体包涵体的细胞。将单个感染沙眼衣原体的细胞收获到预先生长有宿主细胞单层的单独孔中。收获细胞中的包涵体成熟，释放的原体感染宿主细胞单层，从而启动单个包涵体来源的衣原体的繁殖。该方法可用于生成沙眼衣原体的微生物克隆以及回收转化体和突变体。分离出的单个感染衣原体的细胞也可以通过各种下游分子分析进行检查，以揭示单个包涵体水平下衣原体复制的未知特征。
