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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.
BD Horizon™ RB780 Mouse Anti-Human Ki-67
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- 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.
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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.
研发参考 (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). 查看参考
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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). 查看参考
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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). 查看参考
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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). 查看参考
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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). 查看参考
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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). 查看参考
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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). 查看参考
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Sun X, Kaufman PD. Ki-67: more than a proliferation marker. Chromosoma. 2018; 127(2):175-186. (Biology). 查看参考
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