Advertisement

Neutrophil pp 117-125 | Cite as

Microinjection and Micropipette-Controlled Phagocytosis Methods for Neutrophils

  • Maurice B. HallettEmail author
  • Jennie S. Campbell
  • Iraj Laffafian
  • Sharon Dewitt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2087)

Abstract

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization.

Key words

Microinjection SLAM Electroinjection Phagocytic delivery Cell signaling 

References

  1. 1.
    Guse AH, Berg I, da Silva CP et al (1997) Ca2+ entry by cyclic ADP-ribose in intact T-lymphocytes. J Biol Chem 272:8546–8550CrossRefGoogle Scholar
  2. 2.
    Hallett MB, Campbell AK (1980) Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes; adhesion, endocytosis or fusion? Biochem J 192:587–596PubMedPubMedCentralGoogle Scholar
  3. 3.
    Gao X, Huang L (1995) Cationic liposome-mediated gene transfer. Gene Ther 2:710–722PubMedGoogle Scholar
  4. 4.
    Hallett MB, Campbell AK (1982) Measurement of changes in cytoplasmic free calcium in fused cell hybrids. Nature 294:155–158CrossRefGoogle Scholar
  5. 5.
    Campbell AK, Hallett MB (1983) Measurement of intracellular calcium ions and oxygen radicals in polymorphonuclear leucocyte-erythrocyte "ghost" hybrids. J Physiol 338:537–550CrossRefGoogle Scholar
  6. 6.
    Laffafian I, Hallett MB (1998) Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells. Biophys J 75:2558–2563CrossRefGoogle Scholar
  7. 7.
    Peters R, Sikorski R (1998) Gentle slam. Science 282:2213–2214CrossRefGoogle Scholar
  8. 8.
    Laffafian I, Hallett MB (2000) Gentle micro-injection for myeloid cells using SLAM. Blood 95:3270–3271CrossRefGoogle Scholar
  9. 9.
    Davies-Cox EV, Laffafian I, Hallett MB (2001) Control of Ca2+ influx in human neutrophils by IP3 binding: differential effects of micro-injected IP3 receptor antagonists. Biochem J 355:139–143CrossRefGoogle Scholar
  10. 10.
    Dewitt S, Laffafian I, Hallett MB (2003) Phagosomal oxidative activity during β2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space. J Cell Sci 116:2857–2865CrossRefGoogle Scholar
  11. 11.
    Zimmermann U (1986) Electrical breakdown, electropermeabilization and electrofusion. Ann Rev Physiol Biochem 105:175–256Google Scholar
  12. 12.
    Haas K, Sin WC, Javaherian A et al (2001) Single-cell electroporation for gene transfer in vivo. Neuron 29:583–591CrossRefGoogle Scholar
  13. 13.
    Haas K, Jensen K, Sin WC et al (2002) Targeted electroporation in Xenopus tadpoles in vivo - from single cells to the entire brain. Differentiation 70:148–154CrossRefGoogle Scholar
  14. 14.
    Bestman JE, Ewald RC, Chiu SL et al (2006) In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc 1:1267–1272CrossRefGoogle Scholar
  15. 15.
    Lewis KJ, Masternam B, Laffafian I (2014) Minimal impact electro-injection of cells undergoing dynamic shape change reveals calpain activation. Biochem Biophys Acta (Mol Cell Biol) 1843:1182–1187CrossRefGoogle Scholar
  16. 16.
    Campbell JS, Hallett MB (2015) Active calpain in phagocytically competent human neutrophils: electroinjection of fluorogenic calpain substrate. Biochem Biophys Res Commun 457:341–346CrossRefGoogle Scholar
  17. 17.
    Dewitt S, Hallett MB (2002) Cytosolic free Ca2+ changes and calpain activation are required for beta integrin-accelerated phagocytosis by human neutrophils. J Cell Biol 159:181–189CrossRefGoogle Scholar
  18. 18.
    Dewitt S, Tian W, Hallett MB (2006) Localised PtdIns(3,4,5)P-3 or PtdIns(3,4)P-2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils. J Cell Sci 119:443–451CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Maurice B. Hallett
    • 1
    Email author
  • Jennie S. Campbell
    • 2
  • Iraj Laffafian
    • 1
  • Sharon Dewitt
    • 3
  1. 1.Neutrophil Signalling Group, School of MedicineCardiff UniversityCardiffUK
  2. 2.Neutrophil Signalling Group, School of DentistryCardiff UniversityCardiffUK
  3. 3.School of BiochemistryUniversity of BristolBristolUK

Personalised recommendations