Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers.
Phagocytosis is an important process of the immune system by which pathogens are internalized and eliminated by phagocytic cells. Upon internalization, the phagosome matures and acidifies while being transported in a centripetal fashion. In this chapter, we describe protocols for simultaneous imaging of phagosomal acidification as well as their spatial manipulation by magnetic tweezers. First, we describe the protocols for functionalization of magnetic microbeads with pH-sensitive dyes and pH calibration of these particles. We also describe the preparation of magnetic tweezers and the calibration of forces that can be generated by these tweezers. We provide details of the design of the custom electrical and optical setup used for simultaneous imaging of phagosomal pH and phagosome's location. Finally, we provide a detailed description of the data analysis methodology.
Cytoskeleton Dynamics Group, I2BC, CNRS, Gif-sur-Yvette, France. shashank.shekhar@yahoo.com.
Shashank
S
Shekhar
Nanobiophysics Group, University of Twente, Enschede, The Netherlands.|Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Vinod
V
Subramaniam
Nanobiophysics Group, University of Twente, Enschede, The Netherlands. j.s.kanger@utwente.nl.
Johannes S
JS
Kanger
D000818:Animals; D001692:Biological Transport; D002138:Calibration; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000378:metabolism*; D008280:Magnetics / Q000379:methods*; D051379:Mice; D008863:Microspheres; D052898:Optical Tweezers; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D013223:Statistics as Topic; D013997:Time Factors
Acidification; Intracellular manipulation; Magnetic tweezers; Phagocytosis; Quantitative biology; pH-sensitive fluorescent dyes