Edwards Andrew M, Massey Ruth C
Department of Biology and Biochemistry, University of Bath.
J Vis Exp. 2011 Mar 21(49):2693. doi: 10.3791/2693.
Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO(2) at 37°C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.
在此，我们将描述我们如何研究细菌病原体金黄色葡萄球菌对人内皮细胞的侵袭。该通用方案实际上可应用于几乎任何可培养细菌的细胞侵袭研究。我们将重点介绍可研究侵袭特定方面的阶段，例如肌动蛋白重排或小窝的作用。宿主细胞在培养瓶中生长，准备使用时接种到含有Thermanox盖玻片的24孔板中。使用盖玻片便于随后从孔中取出细胞，以减少沉积在孔壁上的血清蛋白（金黄色葡萄球菌会附着于此）的干扰。将细菌培养至所需密度并洗涤以去除任何分泌的蛋白质（例如毒素）。在内皮细胞汇合层的盖玻片在添加细菌之前转移到含有新鲜培养基的新24孔板中。然后将细菌和细胞在37°C、5%二氧化碳环境中一起孵育所需时间。对于金黄色葡萄球菌，这通常在15至90分钟之间。从每个孔中取出Thermanox盖玻片，在PBS中浸洗以去除未附着的细菌。如果要对总相关细菌（粘附和内化的）进行定量，然后将盖玻片放入含有0.5% Triton X-100的PBS的新鲜孔中。轻轻吹打导致细胞完全裂解，通过连续稀释并接种到琼脂平板上对细菌进行计数。如果需要已侵入细胞的细菌数量，将盖玻片添加到含有补充庆大霉素的500 μl组织培养基的孔中，并继续孵育1小时，这将杀死所有外部细菌。然后可以洗涤盖玻片，裂解细胞，并如上所述通过接种到琼脂平板上对细菌进行计数。如果实验需要直接观察，可以固定盖玻片并进行染色以用于光学、荧光或共聚焦显微镜检查，或制备用于电子显微镜检查。
