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Flow cytometric analysis of Bcl-6 expression in Ramos cells. Cells from the human Ramos (Burkitt's lymphoma, ATCC CRL-1596) cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) followed by intracellular staining with either BD Horizon™ R718 Mouse IgG1, κ Isotype Control (Cat. No. 566928; dashed line histogram) or BD Horizon™ R718 Mouse Anti-Human Bcl-6 antibody (Cat. No. 566979/566993; solid line histogram). A fluorescence histogram showing Bcl-6 expression (or Ig Isotype control staining) was derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.
BD Horizon™ R718 Mouse Anti-Bcl-6
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Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
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
- 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.
- 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.
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Companion Products
The K112-91 monoclonal antibody specifically binds to Bcl-6. Bcl-6 was first identified as a proto-oncogene frequently deregulated by chromosomal translocations in non-Hodgkin B-cell lymphomas. It is a nuclear transcriptional repressor of the BTB/POZ zinc-finger family of transcription factors. In addition to its roles in cancer, Bcl-6 plays important roles in the differentiation of normal cells including B cells, thymocytes, CD4+ or CD8+ T cells. Bcl-6 is highly expressed in germinal center B cells, where it promotes the germinal center reaction by inducing proliferation and inhibiting the DNA-damage response. Bcl-6 has been identified as a key factor in promoting the differentiation of CD4+ follicular T helper (Tfh) cells that are involved in promoting germinal center formation and providing help to B cells. The interplay of Bcl-6 and another transcriptional repressor, Blimp-1, is thought to be critical in defining the results of both B-cell and T-cell differentiation.
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 (10)
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Baumjohann D, Okada T, Ansel KM. Cutting Edge: Distinct Waves of BCL6 Expression during T Follicular Helper Cell Development. J Immunol. 2011; 187(5):2089-2092. (Clone-specific: Flow cytometry). View Reference
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Chung Y, Tanaka S, Chu F, et al. Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med. 2011; 17(8):983-988. (Clone-specific: Flow cytometry). View Reference
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Crotty S, Choi YS, Kageyama R, et al. ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6. Immunity. 2011; 34:1-15. (Clone-specific: Flow cytometry). View Reference
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Crotty S, Johnston RJ, Schoenberger SP. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Nat Immunol. 2010; 11(2):114-120. (Biology). View Reference
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Crotty S. Follicular Helper CD4 T Cells (Tfh). Annu Rev Immunol. 2011; 29(1):621-663. (Biology). View Reference
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Eto, D., C. Lao, et al. IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation. PLoS ONE. 2011; 6(3):e17739. (Clone-specific: Flow cytometry). View Reference
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Fazilleau N, McHeyzer-Williams LJ, Rosen H, McHeyzer-Williams MG. The function of follicular helper T cells is regulated by the strength of T cell antigen receptor binding. Nat Rev Immunol. 2009; 10(4):375-384. (Biology). View Reference
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Johnston RJ, Poholek AC, DiToro D, et al. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.. Science. 2009; 325(5943):1006-10. (Biology). View Reference
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Klein U, Dalla-Favera R. Germinal centres: role in B-cell physiology and malignancy. Nat Rev Immunol. 2008; 8(1):22-33. (Biology). View Reference
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Nurieva RI, Chung Y, Martinez GJ, et al. Bcl6 mediates the development of T follicular helper cells. Science. 2009; 325(5943):1001-1005. (Biology). View Reference
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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.
Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.