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Measuring Leukocyte Migration to Nucleotides

  • Taylor J. Moon
  • Michael R. ElliottEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)

Abstract

Extracellular nucleotides are potent damage-associated molecular patterns that shape the immune response to cell stress and tissue damage. These nucleotides are sensed by purinergic receptors and mediate a wide range of cellular effects. Among the best characterized of these effects is cellular migration. While the motility responses of leukocytes to nucleotides can be achieved by microscopic live-cell imaging approaches, such systems are time-consuming and require costly equipment and analysis tools not readily available to all researchers. Transwell migration chambers are a widely used alternative to microscopy due to their relatively low cost and moderate through-put capacity. However, extracellular nucleotides are labile and rapidly degraded in serum-containing cell cultures due to the presence of phosphohydrolases. Thus, evaluating leukocyte migration to nucleotides presents a number of challenges not seen with more stable classes of chemoattractants like proteins and lipids. Here we describe a method to measure leukocyte migration to nucleotides that is cost-effective, rapid and produces robust and reproducible migration of leukocytes using transwell migration chambers.

Key words

Cell migration Chemotaxis Nucleotides ATP UTP Transwell Boyden chamber Leukocytes Purinergic signaling 

Notes

Acknowledgments

This work was supported by NIH grants R01 AI114554, P30 AI027767, and T32 AI049815.

References

  1. 1.
    Zigmond SH, Foxman EF, Segall JE (2001) Chemotaxis assays for eukaryotic cells. Curr Protoc Cell Biol. Chapter 12:Unit 12.1–12.1.29Google Scholar
  2. 2.
    Cekic C, Linden J (2016) Purinergic regulation of the immune system. Nat Rev Immunol 16:177–192CrossRefGoogle Scholar
  3. 3.
    Ferrari D, McNamee EN, Idzko M, Gambari R, Eltzschig HK (2016) Purinergic signaling during immune cell trafficking. Trends Immunol 37:399–411CrossRefGoogle Scholar
  4. 4.
    Burnstock G, Boeynaems J-M (2014) Purinergic signalling and immune cells. Purinergic Signal 10:529–564CrossRefGoogle Scholar
  5. 5.
    Idzko M, Ferrari D, Eltzschig HK (2014) Nucleotide signalling during inflammation. Nature 509:310–317CrossRefGoogle Scholar
  6. 6.
    Medina CB, Ravichandran KS (2016) Do not let death do us part: “find-me” signals in communication between dying cells and the phagocytes. Cell Death Differ 23:979–989CrossRefGoogle Scholar
  7. 7.
    Antonioli L, Pacher P, Vizi ES, Haskó G (2013) CD39 and CD73 in immunity and inflammation. Trends Mol Med 19:355–367CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.David H. Smith Center for Vaccine Biology and ImmunologyUniversity of RochesterRochesterUSA
  2. 2.Department of Microbiology and ImmunologyUniversity of RochesterRochesterUSA

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