R&D Systems
Mouse IL-33 PE antibody
IC3626P
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The aim of this project was to track IL-33 production in mast cells. A directly PE-conjugated anti-mouse IL-33 antibody from R&D Systems has been documented to work in flow cytometry in the company literature and in scientific publications in mouse mast cells. Mast cells markedly upregulate IL-33 by qPCR and ELISA when stimulated by IgE+Antigen or PMA+Ionomycin. Intracellular staining with the IL-33 antibody revealed a pattern of fluorescence consistent with the observed qPCR and ELISA data, with a two-log signal over unstained or isotype control-stained cells. However, in comparison with validated IL-33-knockout cells, the basal one-log shift in fluorescence in unstimulated cells was determined to be non-specific. The antibody therefore should not be used without simultaneous analysis of IL-33-deficient control cells to establish the correct baseline.
Flow Cytometry
Mouse bone marrow-derived mast cells
2x10^5 cells stained after fixation and permeabilization for 30min at 4C with 2ul antibody.
FACS buffer (0.01% NaN3, 2.5% FCS) plus Fc block (clone 93, Biolegend, 1ul/sample).
None
BD FACSCanto LSR flow cytometer
Large background shift in both WT and IL-33-knockout cells demonstrating lack of specificity in staining. Inducible staining following stimulation may be specific (not yet tested). Intensity of staining is not affected by incubation with brefeldin A (prevents secretion), suggesting either lack of specificity or that IL-33 is not secreted (it lacks a secretion signal).
Strong signal in flow cytometry
Signal is mostly non-specific
Not useful without IL-33-deficient cells to establish background.