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      Accutase™ Cell Detachment Solution

      BD™ Accutase™ Cell Detachment Solution

      (RUO)
      Accutase™ Cell Detachment Solution
      Human  Embryonic Stem (ES) cells detached with Accutase™ and analyzed with cell surface markers of pluripotency. H9 ES cells (WiCell, Madison, WI) that were grown on BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277) in mTeSR®1 medium (Stem Cell Technologies) were detached with Accutase™ Cell Detachment Solution. The cells were stained with either PE Mouse anti-SSEA-4 (Cat. 560128) and Alexa Fluor® 647 Mouse anti-Human TRA-1-81 (Cat. 560793) or PE Mouse IgG3, κ Isotype Control (Cat. 556659) and Alexa Fluor® 647 Mouse IgM, κ Isotype Control (Cat. 560806) and then analyzed with Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, Cat. No. D-9542) for live/dead cell discrimination. Flow cytometry was performed on a BD LSR™ II flow cytometry system. For data analysis, live cells were first gated (left panel), and then single cells were selected by light scatter gating (center panel). Expression of surface pluripotency markers was then determined (right panel), with the positions of the quadrant markers based upon the isotype controls (data not shown).
      Accutase™ Cell Detachment Solution
      Human  Embryonic Stem (ES) cells detached with Accutase™ and analyzed with cell surface markers of pluripotency. H9 ES cells (WiCell, Madison, WI) that were grown on BD Matrigel™ hESC-qualified Matrix (Cat. No. 354277) in mTeSR®1 medium (Stem Cell Technologies) were detached with Accutase™ Cell Detachment Solution. The cells were stained with either PE Mouse anti-SSEA-4 (Cat. 560128) and Alexa Fluor® 647 Mouse anti-Human TRA-1-81 (Cat. 560793) or PE Mouse IgG3, κ Isotype Control (Cat. 556659) and Alexa Fluor® 647 Mouse IgM, κ Isotype Control (Cat. 560806) and then analyzed with Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma, Cat. No. D-9542) for live/dead cell discrimination. Flow cytometry was performed on a BD LSR™ II flow cytometry system. For data analysis, live cells were first gated (left panel), and then single cells were selected by light scatter gating (center panel). Expression of surface pluripotency markers was then determined (right panel), with the positions of the quadrant markers based upon the isotype controls (data not shown).
      Product Details
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      Description

      Accutase™ is a cell detachment solution comprised of collagenolytic and proteolytic enzymes and does not contain mammalian or bacterial derived products. Accutase™ is a replacement for trypsin solution and can be used for the passaging of cell lines. Aditionally, Accutase™ performs well when detaching cells for the analysis of many cell surface markers using flow cytometry and for cell sorting. Accutase™ has been demonstrated effective in detaching multiple cell types including: human and mouse embryonic and induced pluripotent stem cells, human neural stem cells, fibroblasts, keratinocytes, vascular endothelial cells, hepatocytes, neural cell types, bone marrow derived stem cells, adherent CHO and BHK cells, macrophages, 293 cells, L929 cells, immortalized mouse testicular germ cells, 3T3, Vero, COS, HeLa, NT2, MG63, M24 and A375 metastatic melanoma, gliomas U251 and D54, HT1080 fibrosarcoma cells, and Sf9 insect cells.

      BD™
      Frozen, sterile aqueous buffered solution containing proprietary ingredients, EDTA, Phenol Red, and no preservative.
      RUO
      AB_2869384
      Cell culture (Routinely Tested), Flow cytometry (Tested During Development)


      Preparation And Storage

      The product should be kept undiluted at -20°C for long term storage, and it may be kept undiluted at 4°C for short term storage.

      This product is shipped frozen, but may thaw during transit. On receipt, if it remains cool to the touch, it may be stored at 4 °C for up to 2 months or re-frozen and stored at -20 °C for up 2 years.

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      Recommended Assay Procedures

      To obtain a single-cell suspension: ( Use aseptic techniques if you plan on continuing culture of cells. )

      1.   Wash the cells with PBS at room temperature.

      2.   Add Accutase™ cell detachment solution to adequately cover the entire area of the culture vessel.

      3.   Incubate at room temperature for 5 to 10 minutes, or until cells are detached. Cells can also be incubated at 37°C, however enzyme activity will decrease over time.

      a.   Additional time and/or Accutase™ may be needed to disassociate three-dimensional structures.

      4.   Triturate the cells to aid in obtaining a single-cell suspension.

      a.   Additional cell culture medium may be added to assist in obtaining a single-cell suspension.

      b.   The additional cell culture medium will neutralize the Accutase™ and allow for passaging of cells without additional washes.

      5.   Remove a small subset of the cell suspension and examine under a microscope to confirm the presence of single cells.

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      Product Notices

      1. Accutase is a registered trademark of Innovative Cell Technologies, Inc.
      2. mTESR™1 is a trademark of StemCell Technologies.
      3. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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      561527 Rev. 2
      Citations & References
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      Development References (5)

      1. Bajpai R, Lesperance J, Kim M, Terskikh AV. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells. Mol Reprod Dev. 2008; 75(5):818-827. (Methodology: Cell culture, Flow cytometry). View Reference
      2. Emre N, Vidal JG, Elia J, et al. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLoS ONE. 2010; 5(8):e12148. (Methodology: Cell culture, Flow cytometry). View Reference
      3. Lamerato-Kozicki AR, Helm KM, Jubala CM, et al. Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation. Exp Hematol. 2006; 34(7):870-878. (Methodology: Cell culture, Flow cytometry). View Reference
      4. Wachs FP, Couillard-Despres S, Engelhardt M, et al. High efficacy of clonal growth and expansion of adult neural stem cells. Lab Invest. 2003; 83(7):949-962. (Methodology: Cell culture, Flow cytometry). View Reference
      5. Weikert C, Eppenberger-Eberhardt M, Eppenberger HM. Cellular engineering of ventricular adult rat cardiomyocytes. Cardiovasc Res. 2003; 59(4):874-882. (Methodology: Cell culture, Flow cytometry). View Reference
      View All (5) View Less
      561527 Rev. 2

      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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