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Multiparameter flow cytometric analysis of HLA-ABC expression on human peripheral blood leucocyte populations. Whole blood was stained with either BD Horizon™ R718 Mouse IgG2a, κ Isotype Control (Cat. No. 566949; Left Plot) or BD Horizon™ R718 Mouse Anti-Human HLA-ABC antibody (Cat. No. 567873/567874; Right Plot). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). A bivariate pseudocolor density plot showing the correlated expression of HLA-ABC (or Ig Isotype control staining) versus side-light scatter (SSC-A) signals was derived from gated events with the forward and side-light scatter characteristics of intact leucocyte populations. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Cell Analyzer System and FlowJo™ software.
BD Horizon™ R718 Mouse Anti-Human HLA-ABC
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Preparation And Storage
Recommended Assay Procedures
BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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).
- An isotype control should be used at the same concentration as the antibody of interest.
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Companion Products
The W6/32 monoclonal antibody recognizes a monomorphic epitope expressed on native β2 microglobulin (β2m)-associated Major Histocompatibility Complex (MHC) class I molecules: Human Leucocyte Antigen (HLA)-A, HLA-B, and HLA-C (HLA-ABC). HLA class I molecules are heterodimers comprised of an ~40-45 kDa, highly polymorphic transmembrane α heavy chain, a type I glycoprotein that is noncovalently-associated with an invariant β2-microglobulin (β2m) light chain. The N-terminal extracellular region of the HLA class I heavy chain is comprised of three domains (α1, α2, and α3). The α1 and α2 domains form a closed antigen-binding groove that accommodates 8-10 aa-peptide antigens. β2m non-covalently associates with the α3 heavy chain domain and promotes HLA class I stability. The W6/32 antibody recognizes a conformational epitope on the HLA class I heavy chain. HLA Class I antigens are normally expressed on most nucleated cells. Their expression is upregulated on activated cells or cells responding to various agents including proinflammatory cytokines or mediators. Reduced HLA Class I expression is found on certain virus-infected or tumor cells. HLA class I antigens expressed on thymic epithelial cells are involved in the positive and negative selection of CD8+ T cell precursors which determines their TCR repertoire during T cell maturation. In the periphery, these HLA Class I antigens serve to either present endogenous antigens or cross-present exogenous antigens for the generation of effector and memory CD8+ T cell responses. Target cell HLA Class I antigens can serve as ligands for inhibitory receptors expressed on NK cells and CD8+ T cells and suppress their cytotoxic responses. Human HLA class I molecules play critical roles in cell-mediated immune responses and tumor surveillance as well as tolerance to self-antigens.
The antibody was conjugated to BD Horizon Red 718, which has been developed exclusively for BD Biosciences as a better alternative to Alexa Fluor® 700. BD Horizon Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor® 700.
Development References (5)
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Barnstable CJ, Bodmer WF, Brown G, et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.. Cell. 1978; 14(1):9-20. (Immunogen: Cytotoxicity, Immunoprecipitation, Radioimmunoassay). View Reference
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Margulies DH, Natarajan K, Rossjohn J, McCluskey J. Major Histocompatibility Complex and Its Proteins. In: Paul WE. Paul WE, ed. Fundamental Immunology 7th Edition. Philadelphia: Lippincott Williams & Wilkins; 2013:487-523.
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Parham P, Barnstable CJ, Bodmer WF. Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C, antigens.. J Immunol. 1979; 123(1):342-9. (Clone-specific: Immunoaffinity chromatography, Immunoprecipitation, Radioimmunoassay). View Reference
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Shields MJ, Ribaudo RK. Mapping of the monoclonal antibody W6/32: sensitivity to the amino terminus of beta2-microglobulin.. Tissue Antigens. 1998; 51(5):567-70. (Clone-specific: Flow cytometry). View Reference
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Wooden SL, Kalb SR, Cotter RJ, Soloski MJ. Cutting edge: HLA-E binds a peptide derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 and inhibits NK cell-mediated lysis.. J Immunol. 2005; 175(3):1383-7. (Clone-specific: Flow cytometry, Functional assay, Inhibition). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.