Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 376, Issue 1-2, Pages 20-31Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2011.10.009
Keywords
T cells; Vasoactive intestinal peptide receptor 1; Vasoactive intestinal peptide receptor 2; Pituitary adenylate cyclase activating; polypeptide-1; Polyclonal antibody; Flow cytometry
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Funding
- NIH/NIDDK [1KO1 DK064828, 2P20 RR015566]
- National Center for Research Resources (NCRR) [P20 RR016741]
- Direct For Biological Sciences
- Div Of Biological Infrastructure [0959512] Funding Source: National Science Foundation
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Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific alpha-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit alpha-VPAC1 polyclonal antibody (alpha-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit a-VPAC1 sera increased the specific-activity of the alpha-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific alpha-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific alpha-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry. (c) 2011 Elsevier B.V. All rights reserved.
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