Kinetic Analysis of Phagosomal ROS Generation
Phagosomal ROS generation is critical for our immune defense against microbial infections. Quantitative assessment of phagosomal ROS production is required to understand the complex relationship between the phagocyte and the microbe, in particular for pathogens that resist phagosomal destruction. ROS detection is difficult due to the transient nature of the reactive species and their multiple interactions with the environment. Direct labeling of phagocytic prey with a ROS-sensitive dye allows to target the dye into the phagosome and to follow the kinetics of phagosomal ROS production on a single phagosome base. Here we describe the basic labeling procedure, the quality assessment, and the imaging technique to achieve this kinetic analysis.
Key wordsNADPH oxidase Phagosome Microbial killing Yeast ROS-sensitive dyes Covalent labeling DCF DCFH2-SE Imaging Flow cytometry
This work was supported by grants from the FRM (Foundation for Medical Research, DCM20121225747). We wish to thank our past colleagues who have contributed to this method and Elodie Hudik for her excellent technical assistance.
- 12.Bernardo J, Long HJ, Simons ER (2010) Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis. Cytom Part A 77A:243–252Google Scholar