Methods for Analyzing Sphingosine-1-Phosphate Signaling in Human and Mouse Primary Mast Cells
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.
KeywordsPrimary mast cells Sphingosine-1-phosphate Signaling Protein phosphorylation Signal transducer and activator of transcription 3 Activation Chemokines Inflammation Western blot Quantitative PCR
Mouse bone marrow-derived mast cell
Bovine serum albumin
Real-time quantitative PCR
Recombinant human stem cell factor
Human skin-derived mast cell
- Stat3 or STAT3
Signal transducer and activator of transcription 3
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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