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Alexa Fluor® 647 Mouse anti-Ki-67
Alexa Fluor® 647 Mouse anti-Ki-67

Immunofluorescent staining of human cell lines.  U-2 OS cells (ATCC HTB-96) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 647 Mouse anti-Ki-67 (Cat. No. 558615; pseudo-colored red, which appears pink when co-localized with the blue) and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 855 Bioimager System with a 20x objective and merged using BD Attovision™ software.  This antibody also stains A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cells, and it works with either cold methanol or Triton X-100 permeabilization (see Recommended Assay Procedure).

Immunofluorescent staining of human cell lines.  U-2 OS cells (ATCC HTB-96) were cultured, fixed, permeabilized with cold methanol, stained with Alexa Fluor® 647 Mouse anti-Ki-67 (Cat. No. 558615; pseudo-colored red, which appears pink when co-localized with the blue) and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure.  The images were captured on a BD Pathway™ 855 Bioimager System with a 20x objective and merged using BD Attovision™ software.  This antibody also stains A549 (ATCC CCL-185) and HeLa (ATCC CCL-2) cells, and it works with either cold methanol or Triton X-100 permeabilization (see Recommended Assay Procedure).

製品詳細
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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
Bioimaging (Routinely Tested), Intracellular staining (flow cytometry) (Tested During Development)
5 µl
AB_647130
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


調製と保管

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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

推奨アッセイ手順

1.        Seed the cells in appropriate culture medium at ~10,000 cells per well in a 96-well Imaging Plate, and

        culture overnight.

2.        Remove the culture medium from the wells, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

3.        Remove the fixative from the wells, and permeabilize the cells using either cold methanol or Triton™ X-100:

a.        Add 100 µl of -20°C 90% methanol or -20°C BD™ Phosflow Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

OR

b.        Add 100 µl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

4.        Remove the permeabilizer, and wash the wells twice with 100 μl of 1× PBS.

5.        Remove the PBS, and block the cells by adding 100 µl of blocking buffer (3% FBS in 1× PBS) or BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well and incubating for 30 minutes at RT.

6.        Remove the blocking buffer, dilute the antibody conjugate 1:10 in blocking buffer or Stain Buffer (FBS), and stain the cells by adding 50 µl of the diluted antibody conjugate to each well and incubating for 1 hour at RT.

7.        Remove the diluted antibody conjugate, and wash the wells three times with 100 μl of 1× PBS.

8.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

9.        View and analyze the cells on an appropriate imaging instrument.

製品通知

  1. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
  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. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  7. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  10. Triton is a trademark of the Dow Chemical Company.
  11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
558615 Rev. 6
抗体の詳細
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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.

558615 Rev. 6
フォーマットの詳細
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558615 Rev.6
引用&参考文献
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開発者向け参考資料 (9)

  1. 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). 参考文献を見る
  2. 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).
  3. 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). 参考文献を見る
  4. 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). 参考文献を見る
  5. 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). 参考文献を見る
  6. 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). 参考文献を見る
  7. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown.. J Cell Physiol. 2000; 182(3):311-22. (Biology). 参考文献を見る
  8. 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).
  9. 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). 参考文献を見る
すべて表示する (9) 表示項目を減らす
558615 Rev. 6

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