Detection of IL‑33 in bEnd.3 Mouse Cell Line by Flow Cytometry. bEnd.3 mouse endothelioma cell line was stained with Rat Anti‑Mouse IL‑33 PE‑conjugated Monoclonal Antibody (Catalog # IC3626P, filled histogram) or isotype control antibody (Catalog # IC006P, open histogram). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.
Preparation and Storage
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Stability & Storage
Protect from light. Do not freeze.
12 months from date of receipt, 2 to 8 °C as supplied.
IL-33, also known as NF-HEV and DVS 27, is a 30 kDa proinflammatory protein that may also regulate gene transcription (1-3). DVS 27 was identifed as a gene that is upregulated in vasospastic cerebral arteries (1). NF-HEV was described as a nuclear factor that is preferentially expressed in the endothelial cells of high endothelial venules relative to endothelial cells from other tissues (2). IL-33 was identified based on sequence and structural homology with IL-1 family cytokines (3). DVS 27, NF-HEV, and IL-33 share 100% amino acid sequence identity. IL-33 is constitutively expressed in smooth muscle and airway epithelia. It is upregulated in arterial smooth muscle, dermal fibroblasts, and keratinocytes following IL-1 alpha or IL-1 beta stimulation (1, 3). Similar to IL-1, IL-33 can be cleaved in vitro by caspase-1, generating an N-terminal fragment that is slightly shorter than the C-terminal fragment (3, 4). The N-terminal portion of full length IL-33 contains a predicted bipartite nuclear localization sequence and a homeodomain-like helix-turn-helix DNA binding domain. By immunofluorescence, full length IL-33 localizes to the nucleus in HUVECs and transfectants (2). The C-terminal fragment, corresponding to mature IL-33, binds and triggers signaling through mast cell IL-1 R4/ST2L, a longtime orphan receptor involved in the augmentation of Th2 cell responses (3, 5-7). A ternary signaling complex is formed by the subsequent association of IL-33 and ST2L with IL-1R AcP (8). Stimulation of Th2 polarized lymphocytes with mature IL-33 in vitro induces IL-5 and IL-13 secretion (3). In vivo administration of mature IL-33 promotes increased production of IL-5, IL-13, IgE, and IgA, as well as splenomegaly and inflammatory infiltration of mucosal tissues (3). Full length and mature mouse IL-33 share approximately 55% and 90% amino acid (aa) sequence identity with human and rat IL-33, respectively. Mouse IL-33 shares less than 25% aa sequence identity with other IL-1 family proteins.
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Entrez Gene IDs:
90865 (Human); 77125 (Mouse)
C9orf26; C9orf26chromosome 9 open reading frame 26 (NF-HEV); DKFZp586H0523; DVS27; DVS27-related protein; IL1F11; IL-1F11; IL33; IL-33; interleukin 33; Interleukin-1 family member 11; interleukin-33; NFHEV; NF-HEV; NF-HEVNFEHEV; Nuclear factor from high endothelial venules; RP11-575C20.2
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Mast cells were cultured from the bone marrow of IL-33-deficient or WT C57BL/6 mice. Genotypes were confirmed by PCR. Cells (5x10^5) were stained for viability, FceRI and c-kit, blocking Fc receptors, then fixed with BD Cytofix/Cytoperm. Cells were permeabilized with BD Permeabilization Buffer, and stained with 2ul anti-mouse IL-33-PE or rat IgG2a-PE isotype control. Cells were washed three times, and data were acquired on a BD FACSCanto LSR flow cytometer, gating on live c-Kit+FceRI+ cells.Fluorescence increases by two orders of magnitude in both WT and IL-33-KO cells following staining with the IL-33-PE antibody relative to the isotype control. I am currently investigating whether there is any specific signal after stimulation of the cells, at which point IL-33 is induced. Staining with the recommended (greater) amount of antibody produces an even greater background shift in unstimulated cells.R&D Systems Technical Service is investigating.