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Analysis of CD22 (pY822) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells (PBMC) were either stimulated (right panel) by cross-linking of surface IgM with purified Goat F(ab')2 Anti-Human IgM (SouthernBiotech) at 37°C for 2 minutes or unstimulated (left panel). The cells were fixed with pre-warmed BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37ºC, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-CD22 (pY822, Cat. No. 560076) and Alexa Fluor® 647 Mouse anti-human CD20 (Cat. No. 558054). For data analysis, lymphocytes were selected by their scatter profile. The data demonstrates that the upregulated phosphorylation is restricted to the stimulated CD20-positive B lymphocytes. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. We have also demonstrated that this antibody conjugate stains mouse CD22 (pY837) in splenocytes that are activated by IgM cross-linking (data not shown).
The specificity of mAb 12a/CD22 was confirmed by western blot analysis using unconjugated antibody at 0.5 µg/ml on lysates from control (lane 1) and anti-IgM-stimulated (lane 2) mouse splenocytes. Mouse CD22 (pY837) is identified as a band of ~140 kDa in the activated splenocytes. Cross-reactivity to human CD22 (pY822) was demonstrated by western blot analysis on lysates from unstimulated and pervanadate-stimulated Daudi Burkitt's lymphoma cells (data not shown).
BD™ Phosflow PE Mouse anti-CD22 (pY822)
BD™ Phosflow PE Mouse anti-CD22 (pY822)
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Preparation And Storage
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
CD22 is a member of the immunoglobulin (Ig) superfamily that contains several extracellular Ig domains and cytoplasmic Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs). It is expressed in the cytoplasm of pro-B and pre-B cells and on the surface of IgD-positive mature B lymphocytes. Differential splicing of the human CD22 gene results in the expression of two isoforms; the full-length CD22β has 7 Ig domains, while CD22α has only 5. CD22β is the predominant isoform in human cells, at both the mRNA and protein levels. Ligands for the extracellular domains of CD22 are sialic acid-linked glycoproteins on the B cell surface. The intracellular ITIM motifs are substrates of src family tyrosine kinases. Signal transduction is initiated by the phosphorylation of tyrosines in the ITIMs of CD22 upon ligation of the B cell receptor (BCR). Tyrosine phosphorylation in ITIM3 of human CD22 or ITIM2 of mouse CD22 is required for recruitment of the protein tyrosine phosphatase PTP1C (SHP-1), which may terminate signal transduction by CD22 and by co-localized receptors, such as the BCR. Studies of mice deficient for CD22 suggest that this adhesion molecule generates both negative and positive regulatory signals that affect B cell development and responsiveness.
The 12a/CD22 monoclonal antibody recognizes the phosphorylated tyrosine 822 (pY822) in ITIM3 of human CD22β. The equivalent site in human CD22α is pY645. The orthologous phosphorylation site in the predominant isoform of mouse CD22 is pY837, which is in ITIM2.
Development References (3)
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Fujimoto M, Kuwano Y, Watanabe R, et al. B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation. J Immunol. 2006; 176:873-879. (Biology).
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Grewal :K, Boton M, Ramirez K, et al. ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling. Mol Cell Biol. 2006; 26(13):4970-4981. (Biology).
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Tedder TF, Tuscano J, Sato S, Kehrl JH. CD22, a B lymphocyte-specific adhesion molecule that regulates antigen receptor signaling. Annu Rev Immunol. 1997; 15:481-504. (Biology).
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