FreeStyle 293 Expression System
Contents
1. Store frozen cells in liquid nitrogen until ready to use To thaw and establish cells 1 Remove the cryovial of cells from the liquid nitrogen and thaw quickly in a 37 C water bath 2 Just before the cells are completely thawed decontaminate the outside of the vial with 70 ethanol Triturate and transfer the entire contents of the cryovial into a 125 ml polycarbonate disposable sterile Erlenmeyer shaker flask containing 17 ml of pre warmed FreeStyle 293 Expression Medium 3 Incubate cells in a 37 C incubator containing a humidified atmosphere of 8 CO in air on an orbital shaker platform rotating at 125 rpm Loosen the cap of the flask a quarter turn from snug to allow oxygenation aeration 4 Once the culture has reached greater than 1 x 10 viable cells ml typically 3 to 5 days transfer the cell suspension aseptically into a centrifuge tube and vortex for 10 seconds 5 Determine viable and total cell counts see protocol on page 4 Subculture the FreeStyle 293 F cells by seeding shaker flasks at 3 x 10 viable cells ml in pre warmed FreeStyle 293 Expression Medium We generally use 125 or 250 ml polycarbonate disposable sterile Erlenmeyer flasks containing 20 or 40 ml total working volume of cell suspension respectively Important Note Subculture cells a minimum of two additional times before use in transfection experiments to allow opportunity for recovery from thawing To subculture cells see the proced2. virus CMV promoter Successful transfection will result in B galactosidase expression that is easily assayed see the next page For a map of pCMV SPORT Bgal see page 12 You may evaluate B galactosidase expression by activity assay using cell free lysates Miller 1972 Invitrogen offers the B Gal Assay Kit Catalog no K1455 01 for fast and easy detection of P galactosidase expression continued on next page Transfecting Cells continued Plasmid Preparation Materials to Have on Hand Optimal Conditions for 30 ml Transfection Note Plasmid DNA for transfection into eukaryotic cells must be clean sterile and free from phenol and sodium chloride Contaminants may kill the cells and salt will interfere with complexing decreasing transfection efficiency We recommend isolating plasmid DNA using one of the Purelink HiPure Plasmid Kits Catalog no K2100 14 or K2100 16 Note Make sure your DNA preparation is sterile for instance by performing filtration through a 0 22 pm filter before use You will need to have the following reagents on hand before beginning e Suspension FreeStyle 293 F cells cultured in FreeStyle 293 Expression Medium Recommendation Calculate the number of cells that you will need for your transfection experiment and expand cells accordingly Make sure that the cells are healthy and greater than 90 viable before proceeding to transfection e Purified plasmid DNA of int
3. Components of Introduction The FreeStyle 293 Expression System is designed to allow large scale transfection of suspension 293 human embryonic kidney cells in a defined serum free medium The system includes FreeStyle 293 F cells that have been adapted to serum free suspension culture in FreeStyle 293 Expression Medium Transfection and expression experiments may be performed directly in FreeStyle 293 Expression Medium without the need to change media The complete FreeStyle 293 Expression Kit provides enough reagents to perform 16 transfections in a 30 ml volume but larger volume transfections may be performed using simple scale up of reagents The FreeStyle 293 Expression System includes the following major components the FreeStyle 293 FreeStyle 293 F cells This cell line is adapted to high density serum free Expression System suspension culture in FreeStyle 293 Expression Medium and is capable of producing high levels of recombinant protein see the next page for more information TM e FreeStyle 293 Expression Medium This is a defined serum free medium formulated specifically to allow growth and large scale transfection of suspension FreeStyle 293 F cells see page 3 for more information e 293fectin This transfection reagent provides high transfection efficiency in suspension FreeStyle 293 F cells FreeStyle 293 F Cells Introduction Parental Cell Line Charact
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5. procedure below to transfect suspension FreeStyle 293 F cells in a 30 ml volume Remember that you may keep the cells in FreeStyle 293 Expression Medium during transfection We recommend including a positive control pCMV SPORT fgal and a negative control no DNA no 293fectin in your experiment 1 The day before transfection day 1 determine the number of cells that you will need for your experiment Remember that for each 30 ml transfection you will need 3 x 10 cells in 28 ml of FreeStyle 293 Expression Medium Tip To transfect cells on day 2 seed cells at a density of 6 7 x 10 viable cells ml To transfect on day 3 seed cells at a density of 3 4 x 10 cells ml 2 On the day of transfection transfer a small aliquot of the cell suspension to a microcentrifuge tube and determine viability and the amount of cell clumping using the trypan blue dye exclusion method Vigorously vortex for 45 seconds to break up cell clumps and determine total cell counts using a Coulter Counter or a hemacytometer Viability of cells must be over 90 Important For optimal transfection results make sure that you have a single cell suspension It may be necessary to vortex the cells for 10 to 30 seconds 3 Calculate the volume of cell suspension containing the number of cells needed for one transfection for each 30 ml transfection you will need 3 x 10 cells Place the shaker flask containing cells in a 37 C incubator on an orbital s
6. x 10 viable cells ml TM e Use a freezing medium composed of 90 fresh FreeStyle 293 Expression Medium and 10 DMSO Guidelines to prepare freezing medium and to freeze cells are provided in this section Preparing Freezing Medium Prepare freezing medium immediately before use 1 Ina sterile conical centrifuge tube mix together the following reagents for every 1 ml of freezing medium needed TM FreeStyle 293 Expression Medium 0 9 ml DMSO 0 1 ml 2 Filter sterilize the freezing medium and place the tube on ice until use Discard any remaining freezing medium after use Freezing Cells Before starting label cryovials and prepare freezing medium Keep the freezing medium on ice 1 Grow the desired quantity of FreeStyle 293 F cells in shaker flasks harvesting when the cell density reaches 0 5 to 1 x 10 viable cells ml Transfer cells to a sterile conical centrifuge tube 2 Determine the viable and total cell counts see protocol on page 4 and calculate the volume of freezing medium required to yield a final cell density of 5 8 x 10 viable cells ml 3 Centrifuge cells at 100 x g for 5 minutes at room temperature and carefully aspirate the medium 4 Resuspend the cells in the pre determined volume of chilled freezing medium 5 Place cryovials in a microcentrifuge rack and aliquot 1 ml of the cell suspension into each cryovial 6 Freeze cells in an automated or manual controlled rate
7. 3 7200 or e mail outlicensing lifetech com Product Qualification Introduction FreeStyle 293 F Cells FreeStyle 293 Expression Medium 293fectin Opti MEM Reduced Serum Medium pCMV SPORT fgal This section describes the criteria used to qualify the components of the FreeStyle 293 Expression System Each lot of cells is tested for cell growth and viability post recovery from cryopreservation Master Cell Banks are screened for viruses mycoplasma and sterility Species identity is confirmed by isozyme and karyotype analysis Each lot of FreeStyle 293 Expression Medium is tested to ensure conformance with the most current approved product specification This currently consists of tests for pH osmolality endotoxin sterility and a three passage 293 Performance Assay For individual lot test results and more information contact Gibco Technical Service at 1 800 828 6686 e 293fectin is tested functionally in a transfection using a luciferase reporter containing plasmid e 293fectin is tested for the absence of microbial contamination using blood agar plates Sabaraud dextrose agar plates and fluid thioglycolate medium e Opti MEM I is subjected to pH osmolality endotoxin bacterial fungal and mycoplasma testing The endotoxin level is less than 1 0 EU mL e Each lot of Opti MEM I is evaluated utilizing sensitive quantitative assays for its ability to support cloning
8. Harbor New York Cold Spring Harbor Laboratory 2002 2007 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 16 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
9. and 1 Transfer a small aliquot of the cell suspension to a microcentrifuge tube iability 2 Determine viability and the amount of cell clumping using the trypan blue dye exclusion method Vigorously vortex for 10 30 seconds to break up cell clumps Determine cell density electronically using a Coulter Counter or manually using a hemacytometer Thawing and Establishing Cells Introduction Materials Needed Thawing Procedure TM Follow the protocol below to thaw FreeStyle 293 F cells to initiate cell culture The FreeStyle 293 F cell line is supplied in a vial containing 1 ml of cells at 1 x 107 viable cells ml in 90 FreeStyle 293 Expression Medium and 10 DMSO Thaw FreeStyle 293 F cells directly into the FreeStyle 293 Expression Medium supplied with the kit You will need to have the following reagents on hand before beginning TM e FreeStyle 293 F cells supplied with the kit store frozen cells in liquid nitrogen until ready to use e FreeStyle 293 Expression Medium supplied with the kit pre warmed Note We do not recommend adding antibiotics to media as this may negatively impact cell growth e 125 ml polycarbonate disposable sterile Erlenmeyer flask e Orbital shaker in 37 C incubator with a humidified atmosphere of 8 CO e Room temperature table top centrifuge and sterile centrifuge tubes e Reagents to determine viable and total cell counts e Sterile 50 ml conical tubes
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11. e following reagents supplied in the FreeStyle 293 Expression System and Products other reagents suitable for use with the kit are available separately from Invitrogen Ordering information is provided below Item Amount Catalog no FreeStyle 293 F Cells 1 vial 1 x 10 cells R790 07 FreeStyle 293 Expression Medium 1L 12338 018 6x1L 12338 026 293fectin 1ml 12347 019 Opti MEM I Reduced Serum 100 ml 31985 062 Medium 1X liquid 500 ml 31985 070 PureLink HiPure Plasmid Midiprep 25 preps K2100 04 Kit PureLink HiPure Plasmid Maxiprep 10 preps K2100 06 Kit PureLink HiPure Plasmid Filter 10 preps K2100 16 Maxiprep Kit PureLink HiPure Plasmid 4 preps K2100 08 Megaprep Kit Trypan Blue Stain 100 ml 15250 061 pCMV SPORT fgal 25 ug 10586 014 B Gal Assay Kit 100 reactions K1455 01 FreeStyle MAX Protein production can also be performed in CHO cells or 293 cells using one of Expression the FreeStyle MAX Expression Systems Below the ordering information is Systems provided for the reagents specific for the FreeStyle MAX Expression Systems Item Amount Catalog no FreeStyle MAX 293 Expression 25 reactions 30 ml K9000 10 System cultures FreeStyle MAX CHO Expression 25 reactions 30 ml K9000 20 System cultures FreeStyle CHO S Cells 1 vial 1 x 10 cells R800 07 FreeStyle CHO Expression Medium 1L 12651 014 6x1L 12651 022 vi Overview Introduction
12. efficiency of a murine myeloma cell line and growth over multiple subcultures of an adherent cell line Test lots of Opti MEM I at 2 CHO growth and 4 Sp2 cloning serum supplementation are compared to a previously approved Opti MEM I control e Gibco cell culture liquid products are prepared by an aseptic process for which each step has been validated to ensure that all products meet the industry standard sterility assurance level of 10 i e product that demonstrates a contamination level of no more than 1 of 1000 units during the manufacturing process The highest level of sterility assurance equal to or greater than 10 cannot be achieved without terminal sterilization which is harmful to the performance of cell culture products The pCMV SPORT Bgal plasmid has passed the following quality control assays e Purity DNA structure and selected restriction sites are verified by agarose gel analysis e Expression of B gal activity is verified by transfection into BHK 21 cells and in situ staining with X gal 15 References Graham F L Smiley J Russell W C and Nairn R 1977 Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5 J Gen Virol 36 59 74 Harrison T Graham F and Williams J 1977 Host range Mutants of Adenovirus Type 5 Defective for Growth in HeLa Cells Virology 77 319 329 Miller J H 1972 Experiments in Molecular Genetics Cold Spring
13. erest e 293fectin supplied with the kit store at 4 C until use e Opti MEM I Reduced Serum Medium supplied with the kit pre warmed e FreeStyle 293 Expression Medium supplied with the kit pre warmed Note Do not add antibiotics to media during transfection as this may decrease transfection activity e 125 ml polycarbonate disposable sterile Erlenmeyer flasks e Orbital shaker in 37 C incubator with a humidified atmosphere of 8 CO e Room temperature table top centrifuge and sterile conical centrifuge tubes e Reagents to determine viable and total cell counts e Sterile disposable polycarbonate snap cap tubes e Vortex mixer We generally perform transfection experiments in a 30 ml volume To transfect suspension FreeStyle 293 F cells we recommend using the following optimized conditions e Final transfection volume 30 ml Number of cells to transfect 3 x 10 cells final cell density of 1 x 10 cells ml e Amount of plasmid DNA 20 40 ug we typically use 30 ug TM e Amount of 293fectin 40 80 ul we typically use 60 ul Use 2 ul 293fectin per 1 pg of plasmid DNA transfected If you are using other 293 cells you may want to test varying amounts of 293fectin e g 30 40 50 60 80 ul with 30 ug plasmid DNA to determine the optimal conditions for transfection continued on next page 9 Transfecting Cells continued Transfection Procedure 10 TM Follow the
14. eristics of FreeStyle 293 F Cells Note The FreeStyle 293 F cell line is supplied with the FreeStyle 293 Expression System and is derived from the 293 cell line see below FreeStyle 293 F cells are adapted to suspension culture in FreeStyle 293 Expression Medium Frozen cells are supplied in and may be thawed directly into FreeStyle 293 Expression Medium see Thawing and Establishing Cells page 5 The 293 cell line is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA Graham et al 1977 Harrison et al 1977 The E1A adenovirus gene is expressed in these cells and participates in transactivation of some viral promoters allowing these cells to produce very high levels of protein The FreeStyle 293 F cell line supplied with the FreeStyle 293 Expression System is a variant of the 293 cell line that has been adapted to suspension growth in FreeStyle 293 Expression Medium The 293 F cell line was obtained from Robert Horlick at Pharmacopeia The FreeStyle 293 F cell line exhibits the following characteristics e Prepared from low passage Master Cell Bank cultures derived from parental 293 F cells that were re cloned by limiting dilution The 293 clonal derived cultures are maintained in serum free conditions for only 30 to 35 total passages e Adapted to high density serum free suspension growth and are maintained in TM F
15. es grow predominantly as single cells TM It is possible to scale up the FreeStyle 293 F cultures in spinner flasks or bioreactors The appropriate spinner or impeller speed and seeding density should be determined and optimized for each system At Invitrogen the optimum spinner speed was 100 130 rpm and 70 100 rpm impeller speed in Celligen stirred tank bioreactors We recommend seeding cells at 3 5 x 10 viable cells ml Note If the split ratio of cells to fresh media is less than 1 2 you may want to spin down the cell suspension and resuspend the cell pellet in fresh pre warmed FreeStyle 293 Expression Medium prior to inoculating the spinner or bioreactor culture Monitor cell viability and the degree of cell clumping Note that extensive cell clumping may reduce transfection efficiency At high stirring speeds i e greater than 130 rpm and or depending on the impeller design you may want to supplement the FreeStyle 293 Expression Medium with additional Pluronic F 68 2 5 5 ml L of 10 Pluronic F 68 Catalog no 24040 to avoid sheer stress in the culture Pluronic is a registered trademark of BASF Corporation Celligen is a registered trademark of New Brunswick Corp Freezing Cells Introduction You may freeze FreeStyle 293 F cells directly in FreeStyle 293 Expression Medium When freezing the FreeStyle 293 F cell line we recommend the following e Freeze cells at a density of 5 8
16. freezing apparatus following standard procedures For ideal cryopreservation the freezing rate should be a decrease of 1 C per minute 7 Transfer frozen vials to liquid nitrogen for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the procedure outlined in Thawing and Establishing Cells page 5 Transfecting Cells Introduction 293fectin Opti MEM Positive Control Assay for galactosidase Activity To transfect suspension FreeStyle 293 F cells you will use the cationic lipid based transfection reagent 293fectin included with the kit Unlike some other serum free media formulations FreeStyle 293 Expression Medium does not inhibit cationic lipid mediated transfection FreeStyle 293 Expression Medium is specifically formulated to allow high efficiency transfection of suspension FreeStyle 293 F cells without the need to change or add media Transient transfection experiments may be performed in a large volume allowing larger scale protein production 293fectin is a proprietary formulation suitable for transfection of nucleic acids into eukaryotic cells In the FreeStyle 293 Expression System use of 293fectin to transfect FreeStyle 293 F cells provides the following advantages TM TM e 293fectin demonstrates high transfection efficiency in suspension FreeStyle 293 F cells cu
17. haker 4 For each transfection sample prepare lipid DNA complexes by performing the following e Dilute 30 ug of plasmid DNA in Opti MEM I to a total volume of 1 ml Mix gently e Dilute 60 ul of 293fectin in Opti MEM I to a total volume of 1 ml Mix gently and incubate for 5 minutes at room temperature Note Longer incubation times may result in decreased activity e After the 5 minute incubation add the diluted DNA to the diluted TM 293fectin to obtain a total volume of 2 ml Mix gently e Incubate for 20 30 minutes at room temperature to allow the DNA TM 293fectin complexes to form 5 While the DNA 293fectin complexes are incubating remove the cell suspension from the incubator and add the appropriate volume of cell suspension see step 3 into each sterile disposable 125 ml Erlenmeyer shaker flask Add fresh pre warmed FreeStyle 293 Expression Medium up to a total volume of 28 ml for a 30 ml transfection 6 After the DNA 293fectin complex incubation is complete add the 2 ml of DNA 293fectin complex to each shaker flask from Step 4 To the negative control flask add 2 ml of Opti MEMP I instead of DNA 293fectin complex Each flask should contain a total volume of 30 ml with a final cell density of approximately 1 x 10 viable cells ml 7 Incubate the cells in a 37 C incubator with a humidified atmosphere of 8 CO in air on an orbital shaker rotating at 125 rpm 8 Harvest cells or
18. invitrogen FreeStyle 293 Expression System For large scale transfection of suspension 293 cells in a defined serum free medium Catalog no K9000 01 Version D 28 October 2010 25 0439 Table of Contents Table f Contents neien ee en A Stiinta iii Kit C ntentsand Storag esseere rran cnstessihscsendankagn ala eon ta costanssansctevbastagucaleteenladsuestelvonchesSesvandoniabes v Introduction nennen 1 OVELVIEWE Ra Sheila IA ANA AAA Neda 1 Freestyle 295 F Cells 2 2 iii 2 FreeStyle 298 Expression Medium ea iia 3 M thodS citan is 4 General Informatioh ii ia Ai a La airis les 4 Thawing and Establishing Celsa r sesiis iein iin E E E E ET ie KEEDA S eTe IE aE 5 S bcu lt ring Cell A A A BRITEN E A AA es 6 Preezing Cells ad eG A Aaa 7 Transtectine Cell iaa id a anti SS ait ate 8 APPEAR 12 pEMV SPORT Peal incita it Bruns rin a A 12 Technical lA A RATE 13 Purchaser Notification ii ie 14 Product Qualification aia 15 AS NO 16 Kit Contents and Storage Shipping Storage FreeStyle 293 F Cells 293fectin FreeStyle 293 Expression Medium Opti MEM Reduced Serum Medium pCMV SPORT fgal TM The components of the FreeStyle 293 Expression System are shipped and should be stored as listed in the table below For more information about the amount supplied and composition of each reagent see below Contents Shipping Storage FreeStyle 293 F Cells Dry ice Liquid nitr
19. ltured in FreeStyle 293 Expression Medium e DNA 293fectin complexes can be added directly to cells in culture medium e Itis not necessary to remove complexes or change or add medium following transfection 293fectin is available separately from Invitrogen see page Error Bookmark not defined for ordering information For more information see our Web site www invitrogen com or call Technical Service see page 13 Opti MEM I Reduced Serum Medium is included with the FreeStyle 293 Expression System to facilitate optimal formation of DNA 293fectin complexes Opti MEM I is a modification of Eagle s Minimal Essential Medium buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine thymidine sodium pyruvate L glutamine trace elements and growth factors The protein level is minimal 15 ug ml with insulin and transferrin being the only protein supplements Phenol red is included at a reduced concentration as a pH indicator Opti MEM I Reduced Serum Medium is available separately from Invitrogen see page Error Bookmark not defined for ordering information For more information see our Web site www invitrogen com or call Technical Service see page 13 pCMV SPORT Bgal is provided as a positive control vector for transfection and expression in FreeStyle 293 F cells The gene encoding galactosidase is expressed in FreeStyle 293 F cells under the control of the human cytomegalo
20. media if recombinant protein is secreted at approximately 48 hours post transfection and assay for recombinant protein expression continued on next page Transfecting Cells continued Optimizing Protein Expression levels may vary depending on the nature of your recombinant protein therefore you may want to perform a time course i e harvest cells or media at 24 Expression Scaling Up Transfections 48 72 96 hours post transfection to optimize expression of your recombinant protein It is possible to perform transfection experiments in a larger e g 1 liter volume If TM you wish to transfect suspension FreeStyle 293 F cells in a larger volume scale up the volume of each reagent accordingly The table below lists suggested conditions to use when transfecting FreeStyle 293 F cells in a 1 liter or 3 8 liter volume The TM optimized conditions to use when transfecting FreeStyle 293 F cells in a 30 ml volume are listed as a reference Note that transfection conditions may vary depending on the type of culture vessel used and the growth conditions of your cells therefore you may want to perform pilot studies to optimize your transfection conditions Transfection Total Amount DNA Dilution Amount of 293fectin Lipid DNA Volume Number of DNA Volume in 293fectin Dilution Volume Complex Volume of Cells Opti MEM I in Opti MEM I 30 ml 3 x 107 30
21. ogen 293fectin Blue ice 4 C FreeStyle 293 Expression Medium Blue ice 2 to 8 C in the dark Opti MEM I Reduced Serum Blue ice 2 to 8 C in the dark Medium pCMV SPORT Bgal Blue ice 20 C Storage conditions Liquid nitrogen Amount supplied One vial containing 1 x 10 cells TM Composition 1 ml of cells in 90 FreeStyle 293 Expression Medium and 10 DMSO Storage conditions 4 C Amount supplied 1 ml sufficient for 16 transfections in a volume of 30 ml using 40 ul of 293fectin per transfection Composition 1 mg ml transfection reagent in membrane filtered water Storage conditions 2 to 8 C in the dark Amount supplied 1 liter Composition Proprietary defined serum free medium formulated with Glutamax I Storage conditions 2 to 8 C in the dark Amount supplied 100 ml Composition Contains HEPES buffer 2 400 mg L sodium bicarbonate hypoxanthine thymidine sodium pyruvate L glutamine trace elements growth factors and phenol red reduced to 1 1 mg L Storage conditions 20 C Amount supplied 25 ug Composition 0 5 ug l in 10 mM Tris HCI pH 7 4 5 mM NaCl 0 1 mM EDTA Accessory Products TM Introduction The products listed in this section may be used with the FreeStyle 293 Expression System For more information refer to our Web site www invitrogen com or call Technical Service see page 13 Accessory Th
22. pports the large scale high density growth of FreeStyle 293 F cells in bioreactors Glutamax I media contain the dipeptide L alanyl L glutamine a stabilized form of L glutamine With Glutamax I media e L glutamine does not degrade in storage or during incubation e Ammonia build up is minimized e Glutamine delivery is controlled e L glutamine does not need to be added at the time of use TM Note Glutamax I is only removed from the medium by cell metabolism There is no accumulation of toxic metabolites due to spontaneous breakdown Typically FreeStyle 293 F cells cultured in FreeStyle 293 Expression Medium demonstrate the following e Doubling time in the range of 20 25 hours Note The doubling time can exceed 25 hours during the first few passages after the cells have been thawed e Cell densities of up to 3 x 10 cells ml in shaker or spinner culture e Cell densities of up to 4 x 10 cells ml in bioreactor culture Note Individual culturing and passaging techniques coupled with cellular heterogeneity inherent within the FreeStyle 293 F cell population may result in experimental variability Methods General Information General Cell Follow the general guidelines below to grow and maintain FreeStyle 293 F cells Handling e All solutions and equipment that come in contact with the cells must be sterile Always use proper sterile technique and work in a laminar flow hood e Befo
23. re starting experiments be sure to have cells established and also have some frozen stocks on hand We recommend using early passage cells for your experiments Upon receipt of the cells from Invitrogen grow and freeze multiple vials of the FreeStyle 293 F cell line to ensure that you have an adequate supply of early passage cells TM e For general maintenance of cells pass FreeStyle 293 F cells when they reach a density of approximately 1 3 x 10 viable cells ml generally every 3 4 days e Use trypan blue exclusion to determine cell viability see below Log phase cultures should be gt 90 viable e When thawing or subculturing cells transfer cells into pre warmed medium TM As with other human cell lines when working with FreeStyle 293 F cells handle as potentially biohazardous material under at least Biosafety Level 2 containment Preparing Media For suspension growth and transfection applications use TM e FreeStyle 293 Expression Medium as is No supplementation is required e Antibiotics are not recommended however 5 ml L of Antibiotic Antimycotic Catalog no 15240 containing penicillin streptomycin and amphotericin B may be used when required TM FreeStyle 293 Expression Medium is extremely sensitive to light For optimal Important results use and store media protected from light Determining Cell Follow the procedure below to determine viable and total cell counts nn
24. reeStyle 293 Expression Medium e Demonstrates high transfection efficiencies with 293fectin e Suspension cultures may be transfected in FreeStyle 293 Expression Medium without the need to change media e Permits transfection of cells at large volumes Other 293 cell lines may be used with the FreeStyle 293 Expression System Before these cell lines may be used for transfection studies however they must be adapted to serum free suspension culture in FreeStyle 293 Expression Medium and evaluated for transfection and expression FreeStyle 293 Expression Medium Introduction Features of the Medium Glutamax I Growth Characteristics of FreeStyle 293 F Cells in the Medium TM FreeStyle 293 Expression Medium is a defined serum free medium specifically developed for the high density suspension culture and transfection of 293 cells The medium contains NO human or animal origin components TM FreeStyle 293 Expression Medium exhibits the following features e An optimized serum free protein free formulation designed to support the high density culture and transfection of 293 cells e g FreeStyle 293 cells in suspension The medium is not recommended for adherent 293 cell culture e Prepared ready to use with no supplementation required e Contains no human or animal origin products e Formulated with Glutamax I see below to increase stability and maximize shelf life e Su
25. rogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or servi
26. ropean Customers Limited Use Label License No 5 Invitrogen Technology 14 Use of the FreeStyle 293 Expression System is covered under a number of different licenses including those detailed below The FreeStyle 293 F Cell Line is genetically modified and carries human adenovirus type 5 DNA As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms 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
27. ug to 1 ml 60 pl to 1 ml 2 ml 1 liter 1x10 1mg to 35 ml 2ml to 35 ml 70 ml 3 8 liter 3 8x 10 3 8 mg to 125 ml 7 6 ml to 125 ml 250 ml Final cell density of 1 x 10 cells ml The transfection efficiency may decrease as the volume increases if the FreeStyle Note 293 F cells are not growing as a single cell suspension i e if significant cell clumping is observed 11 Appendix pCMV SPORT fgal Description pCMV SPORT fgal is included in the FreeStyle 293 Expression System for use as a transfection and expression control and contains the lacZ gene cloned into pCMV SPORT1 The plasmid uses the human cytomegalovirus CMV promoter to control expression of B galactosidase The complete sequence of pCMV SPORT fgal is available for downloading from our Web site www invitrogen com or by calling Technical Service see page 13 Comments for pCMV SPORT fgal 7854 nucleotides SV40 small T intron and polyA signal bases 193 555 complementary strand T7 promoter bases 645 664 lacZ ORF bases 1009 4149 complementary strand SP6 promoter bases 4259 4278 complementary strand CMV promoter bases 4308 4901 complementary strand pUC origin bases 5390 6063 complementary strand loxP bases 6115 6148 Ampicillin bla resistance gene bases 6250 7110 complementary strand incA bases 7134 7306 f1 intergenic region bases 7579 7854 complementary strand 12 Technical Service Web Resources Visit the Invit
28. ure on the next page Subculturing Cells Passaging Cells ur RECO Scaling Up Cell Culture Noirs N END Y Subculture cells when the density is approximately 2 3 x 10 viable cells ml typically every 3 4 days When maintaining FreeStyle 293 F cells we generally use a 125 or 250 ml polycarbonate disposable sterile Erlenmeyer flask containing 25 to 40 ml or 50 to 80 ml total working volume of cell suspension respectively Note Glass flasks without baffles may be used but thorough cleaning after each use is essential to avoid potential toxicity which is more problematic in serum free cultures 1 Determine viable and total cell counts see protocol on page 4 2 Using the cell density determined in Step 1 calculate the split ratio needed to seed the new shaker flask at 3 x 10 viable cells ml 3 Dilute the cells in fresh pre warmed FreeStyle 293 Expression Medium to give a final cell density of 0 1 0 2 x 10 viable cells ml in the desired final volume 4 Incubate flasks in a 37 C incubator containing a humidified atmosphere of 8 CO in air on an orbital shaker platform rotating at 135 rpm 5 Repeat Steps 1 5 as necessary to maintain or expand cells Monitor the degree of cell clumping see below TM FreeStyle 293 F suspension cultures may grow as 2 to 10 cell clusters Vigorous vortexing for 10 30 seconds may be required at each subculture for a number of passages until the cultur
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