Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers

Part of the Methods in Molecular Biology book series (MIMB, volume 1519)


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 


  1. 1.
    Flannagan RS, Jaumouille V, Grinstein S (2012) The cell biology of phagocytosis. Annu Rev Pathol 7:61–98. doi: 10.1146/annurev-pathol-011811-132445 CrossRefPubMedGoogle Scholar
  2. 2.
    Heinrich V (2015) Controlled one-on-one encounters between immune cells and microbes reveal mechanisms of phagocytosis. Biophys J 109(3):469–476. doi: 10.1016/j.bpj.2015.06.042 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Toyohara A, Inaba K (1989) Transport of phagosomes in mouse peritoneal macrophages. J Cell Sci 94(Pt 1):143–153PubMedGoogle Scholar
  4. 4.
    Harrison RE, Bucci C, Vieira OV, Schroer TA, Grinstein S (2003) Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP. Mol Cell Biol 23(18):6494–6506CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Blocker A, Severin FF, Burkhardt JK, Bingham JB, Yu H, Olivo JC, Schroer TA, Hyman AA, Griffiths G (1997) Molecular requirements for bi-directional movement of phagosomes along microtubules. J Cell Biol 137(1):113–129CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Falcon-Perez JM, Nazarian R, Sabatti C, Dell'Angelica EC (2005) Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J Cell Sci 118(Pt 22):5243–5255CrossRefPubMedGoogle Scholar
  7. 7.
    Diwu Z, Chen CS, Zhang C, Klaubert DH, Haugland RP (1999) A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells. Chem Biol 6(7):411–418CrossRefPubMedGoogle Scholar
  8. 8.
    VonSteyern FV, Josefsson JO, Tagerud S (1996) Rhodamine B, a fluorescent probe for acidic organelles in denervated skeletal muscle. Journal of Histochemistry & Cytochemistry 44(3):267–274CrossRefGoogle Scholar
  9. 9.
    Gruenberg J, Griffiths G, Howell KE (1989) Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol 108(4):1301–1316CrossRefPubMedGoogle Scholar
  10. 10.
    Matteoni R, Kreis TE (1987) Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol 105(3):1253–1265CrossRefPubMedGoogle Scholar
  11. 11.
    Shekhar S, Cambi A, Figdor CG, Subramaniam V, Kanger JS (2012) A method for spatially resolved local intracellular mechanochemical sensing and organelle manipulation. Biophys J 103(3):395–404. doi: 10.1016/j.bpj.2012.06.010 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Shekhar S, Klaver A, Figdor CG, Subramaniam V, Kanger JS (2010) Spatially resolved local intracellular chemical sensing using magnetic particles. Sensors and Actuators B-Chemical 148(2):531–538CrossRefGoogle Scholar
  13. 13.
    Desjardins M, Griffiths G (2003) Phagocytosis: latex leads the way. Curr Opin Cell Biol 15(4):498–503CrossRefPubMedGoogle Scholar
  14. 14.
    Irmscher M, de Jong AM, Kress H, Prins MW (2013) A method for time-resolved measurements of the mechanics of phagocytic cups. Journal of the Royal Society, Interface / the Royal Society 10 (82):20121048. doi:10.1098/rsif.2012.1048Google Scholar
  15. 15.
    Tanase M, Biais N, Sheetz M (2007) Magnetic tweezers in cell biology. Methods Cell Biol 83:473–493CrossRefPubMedGoogle Scholar
  16. 16.
    Bausch AR, Moller W, Sackmann E (1999) Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys J 76(1 Pt 1):573–579CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kanger JS, Subramaniam V, van Driel R (2008) Intracellular manipulation of chromatin using magnetic nanoparticles. Chromosome Res 16(3):511–522. doi: 10.1007/s10577-008-1239-1 CrossRefPubMedGoogle Scholar
  18. 18.
    de Vries AH (2004) High force magnetic tweezers for molecular manipulation inside living cells. University of Twente, Enschede, The NetherlandsGoogle Scholar
  19. 19.
    Dawson RMC, Elliot DC, Elliot WH, Jones KM (1986) Data for Biochemical Research. 3rd ed. edn. Oxford Science Publ.,Google Scholar
  20. 20.
    Hermanson GT (2008) Bioconjugate techniques. Academic, San DiegoGoogle Scholar
  21. 21.
    Yeung T, Terebiznik M, Yu L, Silvius J, Abidi WM, Philips M, Levine T, Kapus A, Grinstein S (2006) Receptor activation alters inner surface potential during phagocytosis. Science 313(5785):347–351CrossRefPubMedGoogle Scholar

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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