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      PE Mouse Anti-Ki-67 Set

      BD Pharmingen™ PE Mouse Anti-Ki-67 Set

      (RUO)
      Product Details
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      BD Pharmingen™
      Human (QC Testing), Mouse (Tested in Development), Rat,Rhesus (Reported)
      Intracellular staining (flow cytometry) (Routinely Tested)
      RUO
      AB_2266296


      Description

      Recognizes Ki-67, a nuclear cell proliferation-associated antigen expressed in all active stages of the cell cycle. Ki-67 is revealed as a double band (345 and 395 kDa) in western blot analysis of proliferating cells. B56 was developed using an immunogen composed of the immunodominant epitope of the Ki-67 protein. Antibodies B56 and MIB 1 react with this immunogen. Flow cytometric analysis reveals that the binding of B56-PE can be blocked by MIB 1 purified antibody.

      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 R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
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      Recommended Assay Procedures

      Ki-67 staining protocol by flow cytometry:

      1.  Harvest, count and pellet cells following standard procedures (Note: Ki-67 is expressed by the proliferative cells. You may get no staining with the resting cells, e.g. unstimulated PBMC).

      2.  While vortexing, add 5 ml drop by drop of cold 70% - 80% ethanol into the cells pellet (1-5 x 10e7 cells). Then incubate at -20°C for 2 hours minimum. These fixed cells can be used up to 60 days after fixing (Store at -20°C).

      3.  Add 30-40 ml wash buffer (PBS with 1% FBS, 0.09% NaN3 pH7.2) to the fixed cells. Centrifuge the cells for 10 minutes at 1000 rpm and aspirate supernatant. Wash one more time with 30-40 ml of wash buffer. Centrifuge at 1000 rpm for 10 minutes and aspirate supernatant.

      4.  Resuspend the cells to a concentration of 1 X 10e7/ ml (1 X 10e6/100 µl).

      5.  Transfer 100 µl cell suspension into each fresh tube.

      6.  Add 20 µl of properly diluted antibody according to the protocol into the tubes above. Mix gently.

      7.  Incubate the tubes at room temperature (RT) for 20-30 minutes in the dark.

      8.  Wash with 2 ml of PBS washing buffer at 1000 rpm for 5 minutes.

      9.  Aspirate the supernatant.

      10. For direct conjugated antibody: go to steps 13 & 14.

      11. For purified antibody: add 50 µl of diluted secondary antibody at optimal concentration (Cat. No. 555988), incubate at RT for 30 minutes in the dark.

      12. Repeat step 8 & 9.

      13. Add 0.5 ml of PBS wash buffer into each tube. For FITC conjugated antibody, add 10 µl of PI Staining Solution (Cat. No. 556463); for PE conjugated antibody, add 20 µl BD Via-Probe™ Cell Viability Solution (Cat. No. 555816) into each tube.

      14. Analyze the sample with FACS.

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      Product Notices

      1. This antibody has been optimized and preassayed with its matched isotype control to be used at the recommended volume of 20 ul/test. Titration of the reagents or substituting with other (non-matched) isotype control is NOT recommended.
      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. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
      5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      556027 Rev. 8
      Components
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      Description Quantity/Size Part Number EntrezGene ID
      PE Mouse Anti-Ki-67 100 Tests (1 ea) 51-36525X N/A
      PE Mouse IgG1, κ Isotype Control 100 Tests (1 ea) 51-35405X N/A
      556027 Rev. 8
      Citations & References
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      Development References (14)

      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. Kubbutat MH, Key G, Duchrow M, Schluter C, Flad HD, Gerdes J. Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein). J Clin Pathol. 1994; 47(6):524-528. (Biology). View Reference
      8. 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
      9. 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
      10. 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
      11. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6):741-748. (Biology). View Reference
      12. 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).
      13. 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
      14. 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
      View All (14) View Less
      556027 Rev. 8

      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.

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