Home

CY-C8250

1. i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Receptor for Advanced Glycation End product RAGE is a multi ligand member of the immunoglobulin superfamily of cell surface molecules that is expressed in a variety of cell lines including endothelial cells smooth muscle cells mononuclear phagocytes pericytes neurons cardiac myocytes mesangial cells and hepatocytes 1 2 RAGE interacts with different structures to transmit a signal into the cell and recognizes three dimensional structures rather than specific amino acid sequences Therefore RAGE seems to fulfill the requirements of a pattern recognition receptor As a member of thesimmunoglobulin superfamily it interacts with a diverse class of ligands including Advanced GlycatiompEnd products AGEs 3 4 HMGBI1 also known as Amphoterin 5 amyloid B peptide 6 amyloid A 7 leukocyte adhesion receptors 8 prions 9 Escherichia coli curli operons 10 B sheet fibrils 11 and several members of the S100 protein superfamily including S100 calgranulins 12 Thus RAGE may have potential involvement in several pathological processes including inflammation diabetes Alzheimer s disease AD systemic amyloidosis and tumor growth 13 RAGE may also mediate physiological functions such as neuronal outgrowth survival and regeneration and play a part in pro inflammatory reactions 12 14 15
2. 000 6 643 651 oO Chavakis T Bierhaus A Al Fakhrti N Schneider D Witte S Linn T Nagashima M Morser J Arnold B Preissner KT et al The pattern recognition receptor RAGE is a counterreceptor for leukocyte integrins a novel pathway for inflammatory cell recruitment J Exp Med 2003 198 1507 1515 O Sasaki N Takeuchi M Ghowei H Kikuchi S Hayashi Y Nakano N Ikeda H Yamagishi S Kitamoto T Saito T et al Advanced glycation end products AGE and their receptor RAGE in the brain of patients with Creutzfeldt Jakob disease with prion plaques Neurosci Lett 2002 326 117 120 10 Chapman MRy Robinson LS Pinkner JS Roth R Heuser J Hammar M Normark S Hultgren SJ Role of Escherichia coli curli operons in directing amyloid fiber formation Science 2002 295 85 1 855 11 Bi rhaus A Humpert PM Morcos M Wendt T Chavakis T Arnold B Stern DM Nawroth PP Understanding RAGE the receptor for advanced glycation end products J Mol Med 2005 83 876 886 12 Hofmann MA Drury S Fu C Qu W Taguchi A Lu Y Avila C Kambham N Bierhaus A Nawroth P et al RAGE mediates a novel proinflammatory axis a central cell surface receptor for 100 calgranulin polypeptides Cell 1999 97 889 901 Cat CY C8250 T Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures 13 Stern DM Yan SD Yan SF and Schmidt AM Receptor for advanced glyc
3. The activation of RAGE initiates nuclear factor kappa B NF B 16 17 and mitogen activated protein kinase MAPK pathways 18 Additionally RAGE mediated cellular stimulation promotes increased expression of the receptor itself This positive feedback loop characterized by ligand receptor interaction followed by increased expression of the receptor suggests that RAGE functions as a propagation and perpetuation factor the two hit model of RAGE engagement is based on this finding 19 Components The RAGE HEK293 Cell Line is supplied in one vial containing approximately 2 x 10 cells in 1 ml of freezing medium complete growth medium w 10 Dimethyl sulfoxide DMSO 1 ml of RAGE HEK293 Cell Line 2 x 10 cells Shipping and Storage The RAGE HEK293 Cell Line is shipped frozen on dry ice It is strongly recommended that the RAGE HEK293 Cell Jine shall be thawed and propagated as soon as possible following receipt see Thawing the RAGE HEK293 Cell Line protocol below If long term storage of the frozen cells is required place vial in the vapor phase of liquid nitrogen Storage of cells directly in liquid nitrogen requires use of protective tubing such as Nunc Cryoflex Tubing Storage of cells at 80 C is suitable only for shortsperiods of time a few months and may result in loss of viability and is not recommended Cat CY C8250 2 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Resear
4. ation endproducts RAGE and the complications of diabetes Ageing Res Rev 2002 1 1 15 14 Gangadharan Sajithlal Henri Huttunen Heikki Rauvala and Gerald M nch Receptor for Advanced Glycation End Products Plays a More Important Role in Cellular Survival than in Neurite Outgrowth during Retinoic Acid induced Differentiation of Neuroblastoma Cells J Biol Chem ge2002 277 6888 6897 15 Henri J Huttunen Carole Fages Juha Kuja Panula Anne J Ridley and Heikki Rauvala Receptor for Advanced Glycation End Products binding COOH terminal Motif of Amphoterin Inhibits Invasive Migration and Metastasis Cancer Res 2002 62 4805 4811 16 Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I et al Diabetesassociated sustained activation of thetranscription factor nuclear factor kappaB Diabetes 2001 50 2792 2808 17 Sousa MM Yan SD Stern D Saraiva MJ Interaction of the receptor for advanced glycation end products RAGE with transthyretin triggers nuclear transcription factor kB NF kB activation Lab Invest 2000 80 1101 1110 18 Degryse B Bonaldi T Scaffidi P Muller S Resnati M Sanvito F Arrigoni G Bianchi ME The high mobility group HMG boxes of the nuclear proteimMHMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells J Cell Biol 2001 152 1197 1206 19 Chavakis T Bierhaus A Nawroth PP RAGE receptor for advanced glycation en
5. ature Transfer entire contents of the vial to the T 75 flask and do not pipette cells up and down as this may kill the cells 4 Swirl the T 75 flask to evenly distribute cells Incubate the flask at 37 C 5 CO overnight to allow cells to attach to the bottom of the flask Nn The following day pour off or aspirate off medium and replace with 15 ml fresh complete medium at room temperature ON Incubate theells at 37 C 5 CO and check daily until they reach 85 95 confluency about 2 4 days Once the cells reach 85 95 confluency subculture the cells as described below For the initial culture it is recommended to archive several frozen stocks and continue to propagate remainder of cells for use in experiments Note Vials inappropriately stored directly in liquid nitrogen without protective tubing such as Nunc Cat CY C8250 3 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Cryoflex Tubing may contain liquid nitrogen Upon thawing the liquid nitrogen will quickly convert to the gas phase and may cause the vial of cells to explode This is a very hazardous situation and should only be performed using protective gloves and clothing and a full face mask To avoid this situation store vials only in the vapor phase of liquid nitrogen or use the protective tubing described above if the vial must be stored directly in th
6. ch Use Only Not for use in diagnostic procedures Safety Guidelines This product contains Dimethyl sulfoxide DMSO a hazardous material It is also important to always follow standard tissue culture practices which include e Wearing gloves safety glasses and a lab coat at all times when conducting the procedure e Carefully performing all procedures to minimize the creation of aerosols or splashes Required Media for RAGE HEK293 Cell Line The list below shows the recommended complete medium and freezing medium fof maintenance of the RAGE HEK293 Cell Line e D MEM high glucose 10 fetal bovine serum FBS e 2 mM L glutamine e 1 Penicillin Streptomycin 10 000 I U Penicillin and 10 000 1g ml Streptomycin e 200 ug ml of G418 Thawing Cells Use the following protocol to thaw the RAGE HEK293 Cell Line to initiate the culture The initial propagation of cells should be used to generate stocks to be frozen and stored for future use 1 Remove the frozen vial of cells from liquid mitrogen and quickly thaw them by swirling in a 37 C water bath Try to keep the O ring and cap ofl the vial out of the water to prevent possible contamination Wear eye protection 2 Before the cells are completely thawed remove from 37 C water bath and decontaminate outside of the vial with 70 ethanol 3 Using sterile techniques transfer th cells to a T 75 cm tissue culture flask containing 15 ml of complete medium at room temper
7. d products a central player in the inflammatory response Microbes Infect 2004 6 1219 1225 Cat CY C8250 8 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Related Products Anti RAGE monoclonal antibody Cat CY M1038 CD36 HEK293 cell line Cat CY C8251 N epsilon Carboxymehtyl Lysine BSA Cat CY R2052 N epsilon Carboxymehtyl Lysine OVA Cat CY R2053 Glucose AGE BSA CY R2056 Glucose AGE OVA CY R2057 Glyceraldehyde AGE BSA Cat CY R2058 Glyceraldehyde AGE OVA Cat CY R2059 Glycolaldehyde AGE BSA Cat CY R2060 Glycolaldehyde AGE OVA Cat CY R2061 Methylglyoxal AGE BSA Cat CY R2062 Methylglyoxal AGE OVA Cat CY R2063 Glyoxal AGE BSA Cat CY R2064 Glyoxal AGE OVA Cat CY R2065 CML HSA N epsilon carboxymethyl Lysine HAS Cat CY R2066 CEL HSA N epsilon carboxyethyl Lysine HAS Cat CY R2067 Human S100B Cat CY R2250 Human S100A6 Cat CY R2256 Human S100A12 Cat CY R2262G Human S100A12 Cat CY R2262H Human S100P Cat CY R2267 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CireuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial pro
8. ducts in tissues Am J Pathol 1993 143 1699 712 U Neeper M Schmidt AM Brett J Yan SD Wang F Pan YC Elliston K Stern D Shaw A Gloning and expression of a cell surface receptor for advanced glycosylation end products of proteins J Biol Chem 1992 267 14998 15004 4 Schmidt AM Vianna M Gerlach M Brett J Ryan J Kao J Esposito C Hegarty H Hurley W Clauss M et al Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface J Biol Chem 1992 267 14987 14997 5 Hori O Brett J Slattery T Cao R Zhang J Chen JX Nagashima M Lundh ER Vijay S Nitecki D et al The receptor for advanced glycation end products RAGE is a cellular binding site for amphoterin Mediation of neurite outgrowth and coexpression of rage and amphoterin in the developing nervous system J Biol Chem 1995 270 25752 2576k an Yan SD Zhu H Fu J Yan SF Roher A Tourtellotte WW Rajavashisth T Chen X Godman GC Stern D et al Amyloidbeta peptide receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage colony stimulating factor a proinflammatory pathway in Alzheimer disease Proc Natl Acad Sci US A 1997 94 5296 5301 N Yan SD Zhu H Zhu A Golabek A DusH Roher A Yu J Soto C Schmidt AM Stern D et al Receptor dependent cell stress and am loid accumulation in systemic amyloidosis Nat Med 2
9. ducts without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY C8250 9 Version 120420
10. e liquid phase of liquid nitrogen Subculturing Cells When the cells reach 85 95 confluency they are ready to be subcultured or transferred to a new tissue culture flask This is typically every 2 3 days Use the following protocol to subculture the cells grown in a T 75 cm flask If a different sized tissue culture flask is being usedjiscalelthe reagent and media volumes accordingly 1 Remove complete medium from the flask by pouring or aspiration Wash th cells once with 5 ml PBS to remove excess medium and discard PBS Complete medium containing FBS will inhibit trypsin 2 Add 5 ml of pre warmed room temperature to 37 C trypsin EDTA 0 5 trypsin with EDTA 2Na solution to the cell monolayer and incubate for 5 minutes at 37 C 5 CO or until cells detach If cells are still attached after 5 minutes swirl the flask gently and incubate a few minutes longer 3 Add 5 ml of complete medium and gently pipette up and down to break up cell clumps and achieve a suspension of single cells Transfer the cell suspension to a 15 ml sterile conical centrifuge tube 4 Determine viable and total cell counts by use of a hemocytometer chamber or a Coulter Counter 5 Dispense 1 ml of the cell suspension into each new T 75 cm flask containing 20 ml of pre warmed medium This is a 1 10 split 1 10 of the originalycell population Cells should be 85 95 confluent after 2 to 3 days If using a culture flask other than a T 75 cm scale
11. ency will yield approximately ten 10 of 1 ml aliquot for freezing 4 Dispense the 1 ml aliquots of the cells into cryovials following manufacturer s recommendations 5 Freeze cells using either a controlled rate freezing apparatus or manually using a freezing container The apparatus should provide a controlled freezing rate of 1 C minute Cells should be frozen to 70 C to 80 C overnight 6 Transfer frozen cell stocks to liquid nitrogen storage the following day Character of RAGE HEK293 Cell Line Fig 1 Western blotting of RAGE HEK293 Cell Line by anti His tag monoclonal antibody 66 lt His tagged RAGE 31 21 5 Cat CY C8250 5 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Fig 2 Flow cytometory analysis of RAGE HEK293 Cell Line by anti RAGE monoclonal antibody YK 2B4 CY M1038 Isotypic IgG Anti RAGE mAb control clone Y K 2B4 Cat CY C8250 6 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures References Neeper M Schmidt AM Brett J et al Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins J Biol Chem 1992 267 14998 5004 2 Brett J Schmidt AM Yan SD et al Survey of the distribution of a newly characterized receptor for advanced glycation end pro
12. i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures Human RAGE expressing HEK293 Cell Line CircuLex RAGE HEK293 Cell Line Cat CY C8250 Intended U S r enaa 1 STOTI od CAE ee eee A 1 Jntroduction 5 5 ccvsictadecustadtosader siaparcnnswensemaeranananue 2 List OF COMmPOD CIS ii sas teastsnscdsnetenucvererseanvonsisvocs 2 Shipping and StOrages sccccasissccascsesccessssedavaseccenntes 2 Safety CUI IME cocsacscussncanatecencedesduneseundennnseanesen 3 Required Media for RAGE HEK293 Cell Line 3 Thawing CMS ceasccsassateendatena shvntesanionssrearbesesasecsnanins 3 Subculturing Cells ccccsceseeeseeeeeeeeeeeeesees 4 Preparation of frozen cell stockS cece 4 5 Character of RAGE HEK293 Cell Line 0000000004 5 6 RRCNSLETICES ccrsaactasdascrabessaciueatnecsstionesiiesdstioenicces Ma 7 8 Related PROUUCLciniansmiprnaihteraiaucnaties 9 Intended Use The CycLex Research Product CircuLex RAGE HEK293 Cell Line was designed for studying RAGE signaling pathway The cell line was derived from HEK293 cell line stably expressing human RAGE which has 6X his tagged at C terminus Applications for this cell line 1 Detecting antibodies or proteinssbinding to RAGE 2 Screening inhibitors of RAGE ligand interaction on cell surface This cell line is for research use only and not for use in diagnostic or therapeutic procedures Cat CY C8250 1 Version 120420
13. the volume of cell suspension used If cells are to be used for an experimental assay seed cells at the required density for the experiment an Incubate the cells at 37 C 5 CO vuntil 85 95 confluent and subculture again or incubate until they reach the desired confluency for the experiment Preparation of frozen cell stocks Before beginning fhe freezing protocol below label all cryovials and prepare freezing medium complete growth medium with 10 DMSO Keep freezing medium at 4 C or on ice until ready for use j Culture a 275m flask of the cells to 85 95 confluency 2 Remove the cells from the flask by following steps 1 through 5 in Subculturing RAGE HEK293 Cell Line above U Centrifuge the remaining cell suspension at 250 x g for 10 minutes at room temperature Aspirate the medium from the cells and resuspend the pelleted cells in 1 ml of freezing medium for every 1 ml of original cell suspension e g if the cells retrieved from the original T 75 cm flask are resuspended in 10 ml and 1 ml is used for subculturing centrifuge the remaining 9 ml of cells aspirate medium and Cat CY C8250 4 Version 120420 i TM RAGE HEK293 Cell Line Circ uL eX User s Manual For Research Use Only Not for use in diagnostic procedures resuspend in 9 ml of freezing medium If more or less cell suspension is used adjust the volume of freezing medium accordingly Each T 75 cm flask at 85 95 conflu

CY-C8250

Contents

Download Pdf Manuals

Related Search

Related Contents