StemPro® Accutase® Cell Dissociation Reagent.pdf

StemPro AccutaseCell Dissociation Reagent StemPro Accutase Cell Dissociation Reagent is a ready-to-use cell detachment solution of proteolytic and collagenolytic enzymes.Useful for the routine detachment of cells from standard tissue culture plasticware and adhesion coated plasticware,including Geltrex Reduced Growth Factor Basement Membrane Matrix,CELLStart and polymers.Accutase performs exceptionally well in detaching cells for the analysis of cell surface markers,virus growth assay,quiescence assays by serum starvation,transformation assays by oncogene transfection,neural crest cell migration assays,cell proliferation,cell haptotaxsis,tumor cell migration assays routine cell passage,production scale-up(bioreactor),and flow cytometry.Cell lines tested for Accutase application includes fibroblasts,keratinocytes,vascular endothelial cells,hepatocytes,vascular smooth muscle cells,hepatocyte progenitors,primary chick embryo neuronal cells,bone marrow 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,D54,HT1080 fibrosarcoma cells,Sf9 insect cells,human embryonic stem cells,human mesenchymal stem cells and human neural stem cells.Accutase does not contain mammalian or bacterial derived products.Description Cat.No.Size StemPro Accutase Cell Dissiciation Reagent A11105-01 1 x 100mL Intended Use For research use only.CAUTION:Not intended for human or animal diagnostic or therapeutic uses.Precautions Do not store Accutase at room temperature.Accutase is stable when stored at 2 to 8C up to 2 years.It is recommended to thaw Accutase at room temperature or 4C overnight.Do not thaw at 37C.Storage Store at-5 to-20C,Protect from light.Shelf Life 24 months Use:Note:The following procedures are designed to dissociate cells on 60mm dish.Volumes should be adjusted accordingly for desired vessel size.General Dissociation:1.Aspirate the medium and wash with 4 mL of DPBS(w/o calcium and magnesium,Cat.No 14190).2.Add Accutase to culture dish or flask using aseptic procedures at 2 mL per 60mm surface area(10 mL per 75cm2 surface area).3.Return culture to 37C incubator and allow cells to detach 5 to 10 minutes.4.Count cells and passage as usual;no additional washes or enzyme inhibitors are required.Dissociation of human ESCs grown in StemPro hESC SFM on Geltrex hESC-qualified or CELLstart coated dishes:1.Aspirate the medium from culture dish and wash with 4 mL of DPBS(w/o calcium and magnesium,Cat.No 14190).2.Aspirate DPBS and add 2 mL of Accutase to culture dish.3.Incubate for 2 to 5 minutes at 37C until individual single cells start to round up.4.Gently rinse to remove cells off of the plates surface.5.Transfer cell suspension to 15 mL conical tube.Gently pipette up and down until cells are in a single cell suspension.6.Add 8 mL of medium to rinse any remaining cells off of the dishs surface and transfer to the conical tube(from Step 5).7.Take a 20 uL sample of the cell suspension to determine viable cell density.8.Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.9.Aspirate supernatant,resuspend in fresh medium and plate on coated dish(s).Incubate at 36 to 38C in a humidified atmosphere of 5%CO2 in air.Note:Plating efficiency of 0.5-1x106 cells/60mm dish is optimal for the culture system of StemPro hESC SFM with Geltrex.Dissociation of adherent human or rat NSCs grown in StemPro NSC SFM on CELLstart coated dishes:1.Aspirate the medium from culture dish and wash with 4 mL of DPBS(w/o calcium and magnesium,Cat.No 14190).2.Aspirate DPBS and add 2 mL of Accutase to culture dish.3.Incubate for 2 to 5 minutes at 37C until individual single cells start to round up.4.Gently rinse to remove cells off of the plates surface.5.Transfer cell suspension to 15 mL conical tube.Gently pipette up and down until cells are in a single cell suspension.6.Add 8 mL of medium to rinse any remaining cells off of the dishs surface and transfer to the conical tube(from Step 5).7.Take a 20 uL sample of the cell suspension to determine viable cell density.8.Centrifuge conical tube containing the cell suspension at 200g for 4 minutes.9.Aspirate supernatant,resuspend in fresh medium and plate on coated dish(s).Incubate at 36 to 38C in a humidified atmosphere of 4 to 6%CO2 in air.Note:Plating efficiency of 1x106 cells/60mm dish is optimal for the culture system of StemPro NSC SFM with CellStart.Dissociation of human or rat neurosphere cultures grown in StemPro NSC SFM:1.Remove neurosphere cell suspension from culture dish and transfer to a 15 mL conical tube.2.Let neurospheres settle down in the tube(2 to 5 minutes)before proceeding to Step 3.Alternatively,the cells can be centrifuged at 100g for 1 minute.3.Gently aspirate medium leaving the neurospheres at the bottom of tube with approximately 100L of media remaining.4.Resuspend neurospheres in 5 mL DPBS(w/o calcium and magnesium,Cat.No 14190).5.Let neurospheres settle down in the tube(2 to 5 minutes)before proceeding to Step 6.Alternatively,the cells can be centrifuged at 100g for 1 minute.6.Gently aspirate DPBS leaving the neurospheres at the bottom of tube with approximately 100L of DPBS remaining.7.Add 1 mL of Accutase to the neurospheres and incubate 10 minutes at room temperature.8.Using the proper sized pipette tip(i.e.1000l),pipette up and down until all the neurospheres are in a single cell suspension.9.Add 4 mL of fresh medium to the tube.10.Centrifuge the cells at 200g for 4 minutes.11.Gently aspirate the supernatant.12.Resuspend cells in fresh medium,transfer to a new culture dish and incubate at 36 to 38C in a humidified atmosphere of 4 to 6%CO2 in air.Note:200,000 cells/mL of cell density can be used for cells in Stempro NSC SFM Related Products StemPro hESC SFM (A1000701)StemPro NSC SFM (A1050901)Geltrex hESC-qualified(A10480)CELLstart(A10142)FGF basic(50ug/mL)(PHG0026)2-Mercaptoethanol(21985)Dulbeccos Phosphate Buffered Saline(DPBS)without calcium,magnesium,or phenol red(1X),liquid(14190)Dulbeccos Phosphate Buffered Saline(DPBS)with calcium,and magnesium(1X),liquid(14040)GLUTAMAXTM-I,200mM(100X),liquid(35050)Technical Support For additional product and technical information,such as Material Safety Data Sheets(MSDS),Certificate of Analysis,etc,please visit our website at .For further assistance,please email our Technical Support team at TechsupportI.GIBCO and STEMPRO are registered trademarks of Life Technologies Corp.CELLstart,Geltrex and GLUTAMAX are trademarks of Life Technologies Corp.Accutase is a registered trademark of Innovative Cell Technologies,Inc.Limited Use Label License No:5 Invitrogen Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer(whether the buyer is an academic or for-profit entity).The buyer cannot sell or otherwise transfer(a)this product(b)its components or(c)materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes.The buyer may transfer information or materials made through the use of this product to a scientific collaborator,provided that such transfer is not for any Commercial Purpose,and that such collaborator agrees in writing(a)not to transfer such materials to any third party,and(b)to use such transferred materials and/or information solely for research and not for Commercial Purposes.Commercial Purposes means any activity by a party for consideration and may include,but is not limited to:(1)use of the product or its components in manufacturing;(2)use of the product or its components to provide a service,information,or data;(3)use of the product or its components for therapeutic,diagnostic or prophylactic purposes;or(4)resale of the product or its components,whether or not such product or its components are resold for use in research.For products that are subject to multiple limited use label licenses,the terms of the most restrictive limited use label license shall control.Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture,use or sale of a therapeutic,clinical diagnostic,vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed,provided that neither this product nor any of its components was used in the manufacture of such product.If the purchaser is not willing to accept the limitations of this limited use statement,Life Technologies is willing to accept return of the product with a full refund.For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing,Life Technologies,5791 Van Allen Way,Carlsbad,California 92008 or References 1.Efficient Propagation of Single Cells Accutase-Dissociated human Embryonic Stem Cells,Bajpai,et al,Journal of Molecular Reproduction and Development,2007,DOI 10.1002/mrd:1-10.2.Human Embryonic Stem Cell-derived Dopaminergic Neurons Reverse Functional Deficit in Parkinsonian Rats,Yang,et al,Stem Cells,2007;0:2007-0494v1.3.Oxygen Reduces Accumulation of Type IV Collagen in Endothelial Cell Subcellular Matrix via Oxidative Stress,T.Brevig,et al,Artificial Organs,Volume 30 Issue 12 Page 915-921,December 2006.4.Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation,Lamerota-Kozicki et al.,Experimental Hematology,Vol.34 Pages 870-878,April 2006.“Adherent cells were detached using 1mM EDTA or Accutase,Preservation of surface antigens(and thus signal strength was better with EDTA,but overall cellular viability was higher with Accutase.”5.The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells,Richards et al.,Journal of Neurochemistry,Vol.97 Page 201,April 2006.6.Nuclear factor-B controls the reaggregation of 3D neurosphere cultures in vitro,Widera et al.,European Cells and Materials,Vol.11,Pages 76-85,2006.7.Rescue Purification Maximizes the Use of Human Islet Preparations for Transplantation,Ichii et al.,American Journal of Transplantation,Vol.5,Pages 21-30,2005.8.Circulating fibroblasts,Phillips,Journal of Clinical Investigation,Vol.114,Pages 438-446,August 2004.9.Cellular engineering of ventricular adult rat cardiomyocytes,Cardiovascular Research,2003 Oct 1;59(4):874-882.“Detachment of cultured vARCs using Accutase is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte grafts.”10.High efficacy of clonal growth and expansion of adult neural stem cells,Lab Investigation.2003 July,Vol.83(7):949-962.“In addition,increased cell survival was obtained when Accutase,instead of trypsin,was used for enzymatic dissociation of NSC cultures.”11.Inhibition of death-receptor mediated apoptosis in human adipocytes by the IGF-1/IGF-1R autocrine circuit,Endocrinology.December 22,2003.12.Binding of gastrointestinal tumor cells to endothelial E-and P-selectin adhesion receptors leads to transient down-regulation of sLeX ligands in vitro,International Journal of Colorectal Disease,Volume 18,Number 4,July 2003,Pages:292 299.13.Adrenomedullin Expression and Growth Inhibitory Effects in Distinct Pulmonary Artery Smooth Muscle Cell Subpopulations,Am.J.Respir.Cell Mol.Biol.,Volume 24,Number 2,February 2001,170-178.March 2009 Form No.5029