Neuenhahn Michael, Schiemann Matthias, Busch Dirk H
Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, München, Germany.
Methods Mol Biol. 2010;595:319-29. doi: 10.1007/978-1-60761-421-0_21.
Direct infection of antigen-presenting cells (APCs) with living intracellular bacteria may influence the early innate immune response as well as the following T-cell response. Revealing the identity of primarily targeted cells during infection is therefore an important task, which requires sensitive methods for the intracellular detection of bacteria before their extensive replication. Determination of colony-forming units (cfu) combines - in contrast to microscopy-based methods - a high sensitivity with the specific recognition of viable multiplying organisms. We recently explored an FACS-based ex vivo cell isolation protocol followed by cfu quantification of lysate-derived bacteria in order to make even very few organisms visible. With this approach, the evaluation of cell-subsets during the earliest phase of bacterial infection became feasible. In general, the assay should be transferable to the ex vivo detection of other intracellular (viral or parasitic) pathogens.
活的细胞内细菌直接感染抗原呈递细胞(APC)可能会影响早期先天免疫反应以及随后的T细胞反应。因此,揭示感染过程中主要靶向细胞的身份是一项重要任务,这需要在细菌大量复制之前对其进行细胞内检测的灵敏方法。与基于显微镜的方法相比,菌落形成单位(cfu)的测定将高灵敏度与对存活繁殖生物体的特异性识别相结合。我们最近探索了一种基于荧光激活细胞分选(FACS)的体外细胞分离方案,随后对裂解物衍生细菌进行cfu定量,以便使即使非常少量的生物体也能被检测到。通过这种方法,在细菌感染的最早阶段对细胞亚群进行评估变得可行。一般来说,该检测方法应可转移用于其他细胞内(病毒或寄生虫)病原体的体外检测。