Methods for Analyzing Sphingosine-1-Phosphate Signaling in Human and Mouse Primary Mast Cells

  • Alena P. Chumanevich
  • Piper A. Wedman
  • Carole A. Oskeritzian
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1697)

Abstract

Mast cells produce a potently bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) constitutively and upon activation. The ligation of S1P to its type 2 receptor on mast cells triggers a novel downstream signaling pathway that we discovered links activation of transcription factor signal transducer and activator of transcription 3 to mast cell-derived chemokine release in both humans and mice. In this chapter, we describe the methods used to study S1P signaling in human and mouse primary mast cells.

Keywords

Primary mast cells Sphingosine-1-phosphate Signaling Protein phosphorylation Signal transducer and activator of transcription 3 Activation Chemokines Inflammation Western blot Quantitative PCR 

Abbreviations

BMMC

Mouse bone marrow-derived mast cell

BSA

Bovine serum albumin

Ct

Threshold cycle

DI

Deionized

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

MC

Mast cell

PBS

Phosphate-buffered saline

p-Stat3

Phosphorylated Stat3

QPCR

Real-time quantitative PCR

rhSCF

Recombinant human stem cell factor

rm

Recombinant murine

S1P

Sphingosine-1-phosphate

S1PR

S1P receptor

SkMC

Human skin-derived mast cell

Stat3 or STAT3

Signal transducer and activator of transcription 3

WB

Western blot

Notes

Acknowledgements

This work was supported by grants from the US National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases R01 AI095494 and NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases R21 AR067996 to C.A.O.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Alena P. Chumanevich
    • 1
  • Piper A. Wedman
    • 1
  • Carole A. Oskeritzian
    • 1
  1. 1.Department of Pathology, Microbiology and ImmunologyUniversity of South Carolina School of MedicineColumbiaUSA

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