TY - JOUR
T1 - Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers
AU - Shekhar, Shashank
AU - Subramaniam, Vinod
AU - Kanger, Johannes S
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Journal Article
UR - http://www.scopus.com/inward/record.url?scp=84994480338&partnerID=8YFLogxK
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U2 - 10.1007/978-1-4939-6581-6_7
DO - 10.1007/978-1-4939-6581-6_7
M3 - Article
C2 - 27815875
SN - 1064-3745
VL - 1519
SP - 93
EP - 112
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -