pp 1-10

Part of the Methods in Molecular Biology book series

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

  • Alena P. Chumanevich
  • Piper A. Wedman
  • Carole A. Oskeritzian
Protocol

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

References

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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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