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563756Image1.png

Two-color flow cytometric analysis of Ki-67 expression by proliferating MOLT-4 and noncycling human peripheral blood mononuclear cells. Proliferating cells from the human MOLT-4 (T lymphoblastic leukemia, ATCC CRL-1582) cell line and noncycling peripheral blood mononuclear cells (PBMC) were fixed and permeabilized with 70% ice cold ethanol. The cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and stained with BD Horizon™ BV786 Mouse Anti-Ki-67 antibody (Cat. No. 563756) according to the BD Biosciences support protocol, "Flow Cytometry Staining Protocol for Detection of Ki-67." The cells were then counterstained with BD Via-Probe™ [Cat. No. 555815/555816; contains 7-Amino-Actinomycin D (7-AAD)] to stain DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of 7-AAD staining versus Ki-67 were derived from gated events with the forward and side light-scatter characteristics of intact MOLT-4 cells (Left Panel) or PBMC (Right Panel). Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

hKI-67_BV786_MOLT-4.png

Two-color flow cytometric analysis of Ki-67 expression by proliferating MOLT-4 and noncycling human peripheral blood mononuclear cells. Proliferating cells from the human MOLT-4 (T lymphoblastic leukemia, ATCC CRL-1582) cell line and noncycling peripheral blood mononuclear cells (PBMC) were fixed and permeabilized with 70% ice cold ethanol. The cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and stained with BD Horizon™ BV786 Mouse Anti-Ki-67 antibody (Cat. No. 563756) according to the BD Biosciences support protocol, "Flow Cytometry Staining Protocol for Detection of Ki-67." The cells were then counterstained with BD Via-Probe™ [Cat. No. 555815/555816; contains 7-Amino-Actinomycin D (7-AAD)] to stain DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of 7-AAD staining versus Ki-67 were derived from gated events with the forward and side light-scatter characteristics of intact MOLT-4 cells (Left Panel) or PBMC (Right Panel). Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

563756Image1.png hKI-67_BV786_MOLT-4.png hKi-67_BV786_PBMC-L.png Amber-Cap_Violet.png

Two-color flow cytometric analysis of Ki-67 expression by proliferating MOLT-4 and noncycling human peripheral blood mononuclear cells. Proliferating cells from the human MOLT-4 (T lymphoblastic leukemia, ATCC CRL-1582) cell line and noncycling peripheral blood mononuclear cells (PBMC) were fixed and permeabilized with 70% ice cold ethanol. The cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and stained with BD Horizon™ BV786 Mouse Anti-Ki-67 antibody (Cat. No. 563756) according to the BD Biosciences support protocol, "Flow Cytometry Staining Protocol for Detection of Ki-67." The cells were then counterstained with BD Via-Probe™ [Cat. No. 555815/555816; contains 7-Amino-Actinomycin D (7-AAD)] to stain DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of 7-AAD staining versus Ki-67 were derived from gated events with the forward and side light-scatter characteristics of intact MOLT-4 cells (Left Panel) or PBMC (Right Panel). Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

Two-color flow cytometric analysis of Ki-67 expression by proliferating MOLT-4 and noncycling human peripheral blood mononuclear cells. Proliferating cells from the human MOLT-4 (T lymphoblastic leukemia, ATCC CRL-1582) cell line and noncycling peripheral blood mononuclear cells (PBMC) were fixed and permeabilized with 70% ice cold ethanol. The cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) and stained with BD Horizon™ BV786 Mouse Anti-Ki-67 antibody (Cat. No. 563756) according to the BD Biosciences support protocol, "Flow Cytometry Staining Protocol for Detection of Ki-67." The cells were then counterstained with BD Via-Probe™ [Cat. No. 555815/555816; contains 7-Amino-Actinomycin D (7-AAD)] to stain DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of 7-AAD staining versus Ki-67 were derived from gated events with the forward and side light-scatter characteristics of intact MOLT-4 cells (Left Panel) or PBMC (Right Panel). Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

Product Details
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BD Horizon™
MKI67; Antigen identified by monoclonal antibody Ki-67; KIA
Human (QC Testing), Mouse (Tested in Development), Rat, Rhesus (Reported)
Mouse IgG1, κ
Human Ki-67
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2732007
Aqueous buffered solution containing BSA 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 BD Horizon™ BV786 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV786 were removed.
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Recommended Assay Procedures

For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment.  Fluorescent dye interactions may cause staining artifacts which may affect data interpretation.  The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

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Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. BD Horizon Brilliant Violet 786 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
  8. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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563756 Rev. 3
Antibody Details
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B56

The B56 monoclonal antibody specifically binds to the Ki-67 antigen that is expressed in the nucleus of cycling cells (G1, S, G2, M cell cycle phases). During the G0 phase, the antigen cannot be detected. During interphase of the cell cycle, it is associated with nucleolar components, and it is on the surface of the chromosomes during M phase. Ki-67 is a large protein having 2 alternatively spliced isoforms, an N-terminal forkhead-associated domain, a C-terminal domain that binds to heterochromatin proteins, and multiple phosphorylation sites, the functions of which are still unclear. Because of the strict association of Ki-67 expression with cell proliferation, anti-Ki-67 antibodies are useful for the identification, quantification, and monitoring of growing cell populations.

The antibody was conjugated to BD Horizon BV786 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 786-nm.  BD Horizon BV786 can be excited by the violet laser and detected in a filter used to detect Cy™7-like dyes (eg, 780/60-nm filter).  

563756 Rev. 3
Format Details
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BV786
The BD Horizon Brilliant Violet™ 786 (BV786) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This tandem fluorochrome is comprised of a BV421 donor with an Ex Max of 407-nm and an acceptor dye with an Em Max at 786-nm.  BV786, driven by BD innovation, is designed to be excited by the violet laser and detected using a filter, centered near 785 nm (e.g. 780/60 nm bandpass filter).  Please ensure that your instrument’s configurations (lasers and filters) are appropriate for this dye.
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BV786
407 nm
786 nm
563756 Rev.3
Citations & References
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View product citations for antibody "563756" on CiteAb

Development References (12)

  1. Benson MJ, Elgueta R, Schpero W, et al. Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals. J Exp Med. 2009; 206(9):2013-2025. (Clone-specific: Flow cytometry). View Reference
  2. Bigley V, Haniffa M, Doulatov S, et al. The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. J Exp Med. 2011; 208(2):227-234. (Clone-specific: Flow cytometry). View Reference
  3. Bruno S, Crissman HA, Bauer KD, Darzynkiewicz Z. Changes in cell nuclei during S phase: progressive chromatin condensation and altered expression of the proliferation-associated nuclear proteins Ki-67, cyclin (PCNA), p105, and p34. Exp Cell Res. 1991; 196(1):99-106. (Biology: Flow cytometry). View Reference
  4. Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif. 1992; 25(1):31-40. (Biology: Flow cytometry). View Reference
  5. Kill IR. Localisation of the Ki-67 antigen within the nucleolus: evidence for a fibrillarin-deficient region of the dense fibrillar component. J Cell Sci. 1996; 109(6):1253-1263. (Biology). View Reference
  6. Kouro T, Medina KL, Oritani K, Kincade PW. Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators. Blood. 2001; 97(9):2708-2715. (Clone-specific: Flow cytometry). View Reference
  7. Picker LJ, Hagen SI, Lum R, et al. Insufficient production and tissue delivery of CD4+ memory T cells in rapidly progressive simian immunodeficiency virus infection. J Exp Med. 2004; 200(10):1299-1314. (Clone-specific: Flow cytometry). View Reference
  8. Pitcher CJ, Hagen SI, Walker JM, et al. Development and homeostasis of T cell memory in rhesus macaque. J Immunol. 2002; 168(1):29-43. (Clone-specific: Flow cytometry). View Reference
  9. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown.. J Cell Physiol. 2000; 182(3):311-22. (Biology). View Reference
  10. Spargo LDJ, Cleland LG, Cockshell MP, Mayrhofer Graham. Recruitment and proliferation of CD4+ T cells in synovium following adoptive transfer of adjuvant-induced arthritis. Int Immunol. 2006; 18(6):897-910. (Clone-specific: Flow cytometry, Immunofluorescence).
  11. Starborg M, Gell K, Brundell E, Höög C. The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J Cell Sci. 1996; 109(1):143-153. (Biology). View Reference
  12. Valenti LM, Mathieu J, Chancerelle Y, et al. High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis. Exp Cell Res. 2005; 306(1):150-167. (Clone-specific: Flow cytometry). View Reference
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563756 Rev. 3

 

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.

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.