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  3. Human B Cell Signaling Flow Cytometry Panel

Human B Cell Signaling Flow Cytometry Panel #31328

Important Ordering Details

Custom Ordering Details: Product is assembled upon order. Please allow up to three business days for your product to be processed.

    Product Information

    Protocol

    Product Description

    The Human B Cell Signaling Flow Cytometry Panel can be used to interrogate activation of human B cells among peripheral blood mononuclear cells (PBMCs).

    CD45 is a pan leukocyte marker. B cells are identified by expression of CD19. Engagement of the B cell receptor (BCR) triggers the tyrosine phosphorylation of CD79A at Tyr182, which is mediated by Src family kinases. Following engagement of the BCR, Syk is recruited to phosphorylated ITAM motifs within the BCR signaling complex, which facilitates Syk activation through phosphorylation of Tyr525/526 within the kinase activation loop.

    Product Usage Information

    B cell receptor activation can be induced through treatment with Goat Anti-Human IgM, F(ab')2 Antibody (Low Endotoxin, Azide-free) #96232. All antibodies in this kit are compatible with the Intracellular Flow Cytometry Kit (Methanol) #13593 and can be used in a single staining mix on fixed and permeabilized cells. Prior to fixation and antibody incubation, we recommend adding a fixable viability dye such as the Ghost Dye Violet 510 Fixable Viability Dye #59863 to enable identification and exclusion of dead cells from analysis.

    For individual product dilutions, refer to panel product datasheet or individual product pages.

    Gating strategy for observing B cell receptor activation in B cell populations: Apply the same gating strategy for both untreated and treated cell populations. If a fixable viability dye was used, then first gate on viable cells. Next, gate on CD45+ immune cells. Viewing the CD45+ population, gate on singlets using appropriate scatter parameters, such as FSC-A vs. FSC-H. Observe CD19 vs. SSC-A and gate on CD19+ B cells. Observe phospho-CD79A (Tyr182) and phospho-Syk (Tyr525/526) within the CD19+ gate.

    Storage

    CD45 (HI30) Mouse mAb (violetFluor 450 Conjugate) is supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH 7.2. CD19 (Intracellular Domain) (D4V4B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate), Phospho-CD79A (Tyr182) (D1B9) Rabbit mAb (Alexa Fluor® 488 Conjugate), and Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (PE Conjugate) are supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4o C. Do not aliquot the antibodies. Protect from light. Do not freeze.

    All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific “Best By” dates for each individual component.

    Specificity / Sensitivity

    Each antibody in the Human B Cell Signaling Flow Cytometry Panel detects endogenous levels of its target protein. CD45 (HI30) Mouse mAb (violetFluor 450 Conjugate) detects an epitope within the extracellular domain. CD19 (Intracellular Domain) (D4V4B) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) detects an epitope within the intracellular domain. Phospho-CD79A (Tyr182) (D1B9) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of human CD79A protein only when phosphorylated at Tyr188. This corresponds to Tyr182 of mouse CD79A protein. Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (PE Conjugate) detects endogenous levels of Syk protein only when phosphorylated at Tyr525/526 of human Syk. It also detects Syk protein when singly phosphorylated at Tyr526 of human Syk. It does not cross-react with other tyrosine-phosphorylated protein tyrosine kinases.


    Species Reactivity:

    Human

    Source / Purification

    Monoclonal antibodies were purified from tissue culture supernatant via affinity chromatography. The purified antibodies were conjugated under optimal conditions, with unreacted dye removed from the preparation.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    For Research Use Only. Not For Use In Diagnostic Procedures.
    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    Alexa Fluor is a registered trademark of Life Technologies Corporation.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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