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Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers

  • Shashank Shekhar
  • Vinod Subramaniam
  • Johannes S. Kanger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1519)

Abstract

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.

Key words

Phagocytosis Magnetic tweezers Intracellular manipulation Acidification pH-sensitive fluorescent dyes Quantitative biology 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Cytoskeleton Dynamics GroupI2BC, CNRSGif-sur-YvetteFrance
  2. 2.Nanobiophysics GroupUniversity of TwenteEnschedeThe Netherlands
  3. 3.Vrije Universiteit AmsterdamAmsterdamThe Netherlands

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