Flow cytometric analysis of Bcl-6 expression in Ramos cells (Left Panel). Human Ramos cells 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 APC-Cy™7 Mouse Anti-Human Bcl-6 antibody (Cat. No. 563581, solid line histogram) or APC-Cy™7 mIgG1, κ Isotype Control (Cat. No. 557873; dashed line histogram). Fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of Bcl-6 expression in mouse B lymphocytes (Middle and Right Panels). BALB/c mouse mesenteric lymph node cells were stained with APC Rat Anti-Mouse B220 (Cat. No. 553092/561880), BD Horizon™ PE-CF594 Rat Anti-Mouse CD4 (Cat. No. 562285/562314), FITC Hamster Anti-Mouse Fas/CD95 (Cat. No. 554257), and BD Horizon™ V450 Rat Anti-Mouse IgD (Cat. No. 560869) antibodies. Cells were washed and fixed with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049). Cells were permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885), followed by intracellular staining with APC-Cy™7 Mouse Anti-Bcl-6 antibody. A two-color flow cytometric dot plot shows the expression of IgD versus Fas/CD95 by B cells identified as CD4-B220+ from gated events with the forward and side light-scatter characteristics of intact lymphocytes (Middle Panel). Germinal center (GC) B cells were identified as Fas/CD95-positive B cells that expressed low levels of IgD (IgDloCD95/Fas+) whereas non-GC B cells primarily expressed intermediate to high levels of IgD and little or no CD95/Fas. Histograms (Right Panel) derived from gated cell subsets show intracellular Bcl-6 staining levels for mouse GC B cells (solid line) and non-GC B cells (dashed line). Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of Bcl-6 expression in Ramos cells (Left Panel). Human Ramos cells 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 APC-Cy™7 Mouse Anti-Human Bcl-6 antibody (Cat. No. 563581, solid line histogram) or APC-Cy™7 mIgG1, κ Isotype Control (Cat. No. 557873; dashed line histogram). Fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
Multicolor flow cytometric analysis of Bcl-6 expression in mouse B lymphocytes (Middle and Right Panels). BALB/c mouse mesenteric lymph node cells were stained with APC Rat Anti-Mouse B220 (Cat. No. 553092/561880), BD Horizon™ PE-CF594 Rat Anti-Mouse CD4 (Cat. No. 562285/562314), FITC Hamster Anti-Mouse Fas/CD95 (Cat. No. 554257), and BD Horizon™ V450 Rat Anti-Mouse IgD (Cat. No. 560869) antibodies. Cells were washed and fixed with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049). Cells were permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885), followed by intracellular staining with APC-Cy™7 Mouse Anti-Bcl-6 antibody. A two-color flow cytometric dot plot shows the expression of IgD versus Fas/CD95 by B cells identified as CD4-B220+ from gated events with the forward and side light-scatter characteristics of intact lymphocytes (Middle Panel). Germinal center (GC) B cells were identified as Fas/CD95-positive B cells that expressed low levels of IgD (IgDloCD95/Fas+) whereas non-GC B cells primarily expressed intermediate to high levels of IgD and little or no CD95/Fas. Histograms (Right Panel) derived from gated cell subsets show intracellular Bcl-6 staining levels for mouse GC B cells (solid line) and non-GC B cells (dashed line). Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
BCL6; B-cell lymphoma 6 protein; LAZ3; Laz-3, ZBTB27, ZNF51
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Human Bcl-6 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2738291
Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with APC-Cy7 under optimum conditions, and unconjugated antibody and free APC-Cy7 were removed.
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.
- 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.
- APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
- APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Perm/Wash Buffer I RUO
Size
125 mL
Cat No.
557885
APC-Cy™7 Mouse IgG1, κ Isotype Control RUO
Size
100 Tests
Cat No.
557873
563581 Rev. 2
Antibody Details
K112-91
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.
563581 Rev. 2
Format Details
APC-Cy7
APC-Cy7 dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of a Allophycocyanin (APC) donor that has excitation maxima (Ex Max) of 651 nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 779 nm. APC-Cy7 can be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 780 nm (e.g., a 760/60 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
APC-Cy7
Red 627-640 nm
651 nm
779 nm
563581 Rev.2
Citations & References
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
563581 Rev. 2
Please refer to Support Documents for Quality Certificates
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.
