Multiparameter flow cytometric analysis of Ki-67 expression in noncycling Human peripheral blood lymphocytes or proliferating Human MOLT-4 cells. Noncycling normal Human peripheral blood mononuclear cells containing lymphocytes (Top Plots) or proliferating cells from the Human MOLT-4 (T lymphoblastic leukemia, ATCC® CRL-1582™) cell line (Bottom Plots) were permeabilized and fixed with 70% ice-cold ethanol. In separate experiments, the normal or MOLT-4 cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656], stained with either BD Horizon™ RB780 Mouse IgG1, κ Isotype Control (Cat. No. 568532; Left Plots) or BD Horizon™RB780 Mouse Anti-Human Ki-67 antibody (Cat. No. 571659/571737; Right Plots) and counterstained with BD Pharmingen™ DAPI Solution (Cat. No. 564907) to stain DNA. Bivariate pseudocolor density plots showing the correlated expression of DNA (DAPI) versus Ki-67 levels (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes or MOLT-4 cells. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ Software.
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Multiparameter flow cytometric analysis of Ki-67 expression in noncycling Human peripheral blood lymphocytes or proliferating Human MOLT-4 cells. Noncycling normal Human peripheral blood mononuclear cells containing lymphocytes (Top Plots) or proliferating cells from the Human MOLT-4 (T lymphoblastic leukemia, ATCC® CRL-1582™) cell line (Bottom Plots) were permeabilized and fixed with 70% ice-cold ethanol. In separate experiments, the normal or MOLT-4 cells were washed twice with BD Pharmingen™ Stain Buffer (FBS) [Cat. No. 554656], stained with either BD Horizon™ RB780 Mouse IgG1, κ Isotype Control (Cat. No. 568532; Left Plots) or BD Horizon™RB780 Mouse Anti-Human Ki-67 antibody (Cat. No. 571659/571737; Right Plots) and counterstained with BD Pharmingen™ DAPI Solution (Cat. No. 564907) to stain DNA. Bivariate pseudocolor density plots showing the correlated expression of DNA (DAPI) versus Ki-67 levels (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes or MOLT-4 cells. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 Cell Analyzer System and FlowJo™ Software.
Product Details
BD Horizon™
Antigen KI-67; Antigen Ki67; KI-67; KI67; MKI67
Human (QC Testing)
Mouse BALB/c IgG1, κ
Human L428 Hodgkin Lymphoma Cell Line Nuclei
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl/test
4288
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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® CompBeads 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).
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
- Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
- Tandem fluorochromes contain both an energy donor and an energy acceptor. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
- For U.S. patents that may apply, see bd.com/patents.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
RB780 Mouse IgG1, κ Isotype Control RUO
Size
50 µg
Cat No.
568532
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DAPI Solution RUO
Size
1 mg
Cat No.
564907
571659 Rev. 1
Antibody Details
Ki-67
The Ki-67 monoclonal antibody specifically recognizes the Ki-67 antigen (also known as KI67, KI-67). Ki-67 is a nonhistone nuclear protein that is encoded by MKI67 (Marker of Proliferation Ki-67). Ki-67 is associated with cellular proliferation and its expression increases as cells enter and progress through the G1, S, G2, and M cell cycle phases. Ki-67 is a large protein having 2 alternatively spliced isoforms. The Ki-67 protein contains an N-terminal forkhead-associated domain, a protein phosphatase 1-binding domain, a large central region of tandem repeats, and a C-terminal leucine/arginine-rich chromatin-binding domain. Ki-67 is required for cell proliferation and serves as useful marker for distinguishing and analyzing quiescent cells (Ki-67-negative cells within the G0 cell cycle phase) from cycling Ki-67-positive cells including cancer cells.
571659 Rev. 1
Format Details
RB780
The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.
RB780
Blue 488 nm
498 nm
781 nm
571659 Rev.1
Citations & References
View product citations for antibody "571659" on CiteAb
Development References (8)
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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). View Reference
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Byeon IJ, Li H, Song H, Gronenborn AM, Tsai MD. Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67.. Nat Struct Mol Biol. 2005; 12(11):987-93. (Biology). View Reference
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Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.. J Immunol. 1984; 133(4):1710-5. (Clone-specific: Immunocytochemistry). View Reference
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Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation.. Int J Cancer. 1983; 31(1):13-20. (Immunogen: Immunohistochemistry). View Reference
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Kim KH, Sederstrom JM. Assaying Cell Cycle Status Using Flow Cytometry.. Curr Protoc Mol Biol. 2015; 111:28.6.1-28.6.11. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Palutke M, KuKuruga D, Tabaczka P. A flow cytometric method for measuring lymphocyte proliferation directly from tissue culture plates using Ki-67 and propidium iodide.. J Immunol Methods. 1987; 105(1):97-105. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Schwarting R, Gerdes J, Niehus J, Jaeschke L, Stein H. Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67.. J Immunol Methods. 1986; 90(1):65-70. (Clone-specific: Intracellular Staining/Flow Cytometry). View Reference
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Sun X, Kaufman PD. Ki-67: more than a proliferation marker. Chromosoma. 2018; 127(2):175-186. (Biology). View Reference
571659 Rev. 1
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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.