The 3E2 antibody reacts with CD54 (ICAM-1), a 95- kDa member of the Ig superfamily found on lymphocytes, vascular endothelium, high endothelial venules, epithelial cells, macrophages, and dendritic cells. ICAM-1 is a ligand for LFA1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Its expression is upregulated upon stimulation by inflammatory mediators such as cytokines and LPS. Studies with mouse Icam1-transfected antigen-presenting cells, with CD54-blocking antibodies, and in CD54-deficient mice indicate that CD54 participates in inflammatory reactions and antigen-specific immune responses. In addition, there is evidence that CD54 is a receptor involved in MHC-non-restricted responses to weakly immunogenic tumor cells. The 3E2 antibody blocks in vitro and in responses. This antibody is routinely tested by flow cytometric analysis. Other applications were tested during antibody development only or reported in the literature. Upregulation of CD54 expression on activated splenic B lymphocytes. Left panel: Naive BALB/c splenocytes were stained with purified 3E2 mAb (open histogram) followed by FITC-conjugated anti-hamster IgG cocktail (Cat. No. 554011, filled and open histograms). Viable resting lymphocytes were gated according to scatter profile and exclusion of 7-AAD (BD Via-Probe™, Cat. No. 555816/555815). The mean fluorescence intensity of the stained lymphocytes is about 7.5 times greater than that of the negative-control lymphocytes. Right panel: 2-day LPS-activated BALB/c splenocytes were stained with purified 3E2 mAb (open histogram) followed by FITC-conjugated anti-hamster IgG (filled and open histograms). Viable B-cell blasts were gated according to scatter profile and exclusion of 7-AAD. The mean fluorescence intensity of the stained blasts is about 15 times greater than that of the negative-control blasts. Flow cytometry was performed on a BD FACScan™ flow cytometry system.
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