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PerCP-Cy™5.5 Mouse anti-Ki-67
PerCP-Cy™5.5 Mouse anti-Ki-67

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 PerCP-Cy™5.5 Mouse anti-Ki-67 antibody (Cat. No. 561284) according to the BD Biosciences support protocol, Flow Cytometry Staining Protocol for Detection of Ki-67. The cells were then counterstained with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, Cat. No. D-9542) to stain double-stranded DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of DAPI (DNA) 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).

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 PerCP-Cy™5.5 Mouse anti-Ki-67 antibody (Cat. No. 561284) according to the BD Biosciences support protocol, Flow Cytometry Staining Protocol for Detection of Ki-67. The cells were then counterstained with 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, Cat. No. D-9542) to stain double-stranded DNA. Two-color flow cytometric dot plots showing the correlated expression patterns of DAPI (DNA) 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).

Product Details
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BD Pharmingen™
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_10611574
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 PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.

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. 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.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. 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.
  5. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
  6. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  7. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
561284 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.

561284 Rev. 3
Format Details
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PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
561284 Rev.3
Citations & References
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Development References (11)

  1. 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
  2. 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
  3. 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
  4. Ho DWY, Fan ST, To J, et al. Selective plasma filtration for treatment of fulminant hepatic failure induced by D-galactosamine in a pig model. Gut. 2002; 50:869-876. (Clone-specific).
  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. 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
  8. Scholzen T, Endl E, Wohlenberg C, et al. The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure.. J Pathol. 2002; 196(2):135-44. (Biology). 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
View All (11) View Less
561284 Rev. 3

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.