Subculturing Cells
Contents
1. Thawing Cells Introduction The 293FT Cell Line is supplied in a vial containing 3 x 10 cells in 1 ml of Freezing Medium Store frozen 293FT cells in liquid nitrogen until ready to use Handle as potentially biohazardous material under at least Biosafety Level 2 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet before handling Thawing Cells Use the following procedure to thaw 293FT cells to initiate cell culture Thaw cells in prewarmed complete medium without Geneticin 1 Remove the vial of frozen cells from liquid nitrogen and thaw quickly in a 37 C water bath Just before the cells are completely thawed decontaminate the outside of the vial with 70 ethanol and transfer the cells to a sterile 15 ml tube containing PBS Briefly centrifuge the cells at 150 200 x g and resuspend the cells in 2 ml complete medium without Geneticin Transfer the cells to T 75 cm flask containing 10 ml of complete medium without Geneticin Incubate the flask overnight at 37 C for allowing the cells to attach to the bottom of the flask The next day aspirate off the medium and replace with fresh complete medium containing 500 ug ml Geneticin Incubate the cells and check them daily until the cells are 80 90 confluent Proceed to Subculturing Cells next page Y IO mn RECO We recommend subculturing cells for a minimum of2. 3 passages after thawing before use in other applications Subculturing Cells Introduction Subculturing Conditions Determining Viability and Cell Density Follow the recommendations and procedures in this section to subculture 293FT cells Maintain cells as adherent monolayer cultures in complete medium containing 500 ug ml Geneticin Use the following recommended conditions to subculture 293FT cells For a procedure to subculture cells see below Parameter Recommended Condition Cell density gt 5 x 10 viable cells ml gt 80 confluent Culture vessel T 75 cm to T 162 cm disposable sterile T flasks Dilute cells in a total working volume of 15 20 ml for T 75 cm flasks and 40 50 ml for T 162 cm flasks Seeding density 2 to 5 x 10 viable cells cm Incubation conditions 37 C incubator with a humidified atmosphere of 5 10 CO in air loosen caps to allow for oxygenation aeration Follow the procedure below to determine viable and total cell counts using the trypan blue exclusion method 1 7 Transfer a small aliquot of the cell suspension to a microcentrifuge tube and dilute the cells such that the total number of cells counted will not be less than 100 or more than 1 000 To 1 ml of the diluted cell suspension add 100 pl Trypan Blue Stain 0 4 solution Gently aspirate to mix Record the dilution factor The dilution factor equals the total volume amoun
3. at the recommended density see table on previous page diluting in pre warmed complete medium containing 500 ug ml Geneticin Incubate flasks as recommended see table on previous page Maintain cells as adherent monolayer cultures in complete medium containing 500 ug ml Geneticin For the transfection protocol you will need 6 x 10 293FT cells for each sample page 8 Freezing Cells Introduction Once you have established the cells we recommend freezing some cells for future use as described below Freeze cells at a density of at least 3 x 10 viable cells ml Use a freezing medium composed of 90 complete medium and 10 DMSO Prepare freezing medium immediately before use Filter sterilize the freezing medium and store at 4 C until use Discard any remaining freezing medium after use Freezing Cells Before starting label cryovials and prepare freezing medium see above Keep the freezing medium on ice 1 2 8 Culture the desired quantity of 293FT cells to 70 90 confluency Remove the cells from the tissue culture flask s following Steps 1 3 Subculturing Cells page 6 Determine viable and total cell counts see procedure on page 5 and calculate the volume of freezing medium required to yield a final cell density of gt 3x 10 cells ml Prepare the required volume of freezing medium see above Centrifuge the cells suspension from Step 2 at 250 x g for 5 minutes ina table top
4. mM MEM Non Essential Amino Acids NEAA 6 mM L glutamine 1 mM MEM Sodium Pyruvate 1 Pen Strep optional D MEM already contains 4 mM L glutamine which is enough to support cell growth of the 293FT Cell Line However since L glutamine slowly decays over time the complete medium needs to be supplemented with 2 mM L glutamine This will ensure that the concentration of L glutamine in complete medium will not get too low over time due to its slow degradation Note 293FT cells grow well in 6 mM L glutamine but higher concentrations of L glutamine may reduce growth Prepare the complete D MEM medium containing 10 FBS supplemented with 0 1 mM MEM Non Essential Amino Acids 1 mM sodium pyruvate and 2 mM L glutamine as described below using reagents Perform all steps in a tissue culture hood under sterile conditions 1 Remove 100 ml D MEM from 1 L D MEM bottle and replace with 100 ml FBS 2 To the bottle of medium add the following 200 mM L Glutamine 100X 10 ml 10 mM MEM Non Essential Amino Acids 100X 10 ml 100 mM MEM Sodium Pyruvate 100X 10 ml Optional Penicillin Streptomycin 100X 10 ml Filter sterilize the medium using 0 45 um filtration device Store the complete medium at 4 C until use The medium is stable for 6 months at 4 C avoid introducing any contamination into the medium 5 To an aliquot of the complete medium add Geneticin to prepare complete medium with 500 pg ml Geneticin
5. neo Description The pCMVSPORT6TAg neo plasmid is derived from pCMVSPORT6 which has been modified to include the following features The neomycin resistance gene for stable selection in mammalian cells Southern amp Berg 1982 Expression of the neomycin resistance gene is controlled by the SV40 early enhancer promoter from which the SV40 origin of replication has been removed The gene encoding the SV40 large T antigen to facilitate optimal virus production e g Invitrogen s ViraPower Lentiviral Expression System and to permit episomal replication of plasmids containing the SV40 early promoter and origin Expression of the SV40 large T antigen is controlled by the human cytomegalovirus CMV promoter Technical Sup Web Resources port Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs 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 Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Tel 1 760 603 7200 For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Japanese Headquarters European Headquarters LOOP X Bldg 6F Inchinnan B
6. al maintenance of cells pass 293FT cells when they are gt 80 confluent Avoid overgrowing cells before passaging Maintain 293FT cells in complete medium containing 500 ug ml Geneticin Use trypan blue exclusion to determine cell viability Log phase cultures should be gt 90 viable When thawing or subculturing cells transfer cells into medium warmed to room temperature Cells should be at the appropriate confluence and at greater than 90 viability prior to transfection see page 8 Be sure to have the following solutions and supplies available 15 ml sterile conical tubes Appropriate sized tissue culture flasks and pipettes Complete medium see page 3 50 mg ml Geneticin Phosphate Buffered Saline PBS Invitrogen Catalog no 10010 023 Reagents for counting cells Trypsin versene EDTA solution or other trypsin solution Freezing Medium see pages 3 and 7 Table top centrifuge Cryovials if needed Continued on next page General Information continued Media for 293FT Cells Note Preparing Medium The table below lists the recommended complete medium freezing medium and antibiotic concentration required to maintain and culture the 293FT Cell Line Note Fetal bovine serum should not be heat inactivated for use with the 293FT Cell Line Complete Medium Antibiotic Freezing Medium D MEM high glucose 500 ug ml 90 complete medium 10 fetal bovine serum FBS Geneticin 10 DMSO 0 1
7. centrifuge at room temperature Carefully aspirate off the medium and resuspend the cell pellet in the pre determined volume of chilled freezing medium Dispense aliquots of this suspension frequently mixing to maintain a homogeneous cell suspension into cryovials according to manufacturer s specifications Freeze cells in an automated controlled rate freezing apparatus or using a manual method following standard procedures For ideal cryopreservation the freezing rate should be a decrease of 1 C per minute Transfer vials to liquid nitrogen 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 Cells page 4 Transfecting Cells Transfection Methods Transient Transfection Generating Stable Cell Lines Note The 293FT Cell Line is generally amenable to transfection using standard methods including calcium phosphate precipitation Chen amp Okayama 1987 Wieler et al 1977 lipid mediated transfection Felgner et al 1989 Felgner amp Ringold 1989 and electroporation Chu et al 1987 Shigekawa amp Dower 1988 We typically use cationic lipid based transfection reagents to transfect 293FT cells Lipofectamine 2000 is recommended but other transfection reagents are suitable Lipofectamine 2000 is available from Invitrogen see page vi for ordering information The 293FT Cell Line
8. earch 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 Phone 760 603 7200 or e mail outlicensing lifetech com 11 References Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Mol Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Felgner P L Holm M and Chan H 1989 Cationic Liposome Media
9. emarks mentioned herein are the property of Life Technologies Corporation or their respective owners 12 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
10. invitrogen by technologies Growth and Maintenance of the 293FT Cell Line Catalog nos R700 07 Rev Date 14 July 2010 Manual part no 25 0504 MAN0000276 Table of Contents Contents and Storages eseti spike itii eri kesane ASSA vate sekten ene nad behin nel v Accessory Erleis ereechen TEES e AE EE EE ado vi Introduction E 1 Product Information n 2 228 2a 2 ee ee 1 zE 2 General Informa tom sau A 2 RE 4 ee E EE 5 Freezing CellS aan ars el ee ee Ee 7 RE GE 8 e TEE 9 Map of PEMVSPORT6TA 2 neo nungen eren lisis 9 EE EE 10 Purchaser IAN Eeer 11 Referentes Nani aaa 12 Contents and Storage 293FT Cell Line The 293FT Cell Line is used for the production of lentiviral stocks The 293FT Cell Line is supplied as one vial containing 3 x 10 frozen cells in 1 ml of Freezing Medium Upon receipt store in liquid nitrogen until use Handle as potentially biohazardous material under at least Biosafety Level 2 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet before handling Accessory Products Accessory Products vi The products listed below may be used with the 293FT Cell Line For more information refer to our Web site www invitrogen com or call Technical Support see page 10 Note Some reagents are available in other sizes Item Amount Catalog no Lipofec
11. may be transiently transfected with any plasmid General guidelines are provided below e Make sure that cells are healthy at the time of plating Overgrowth of cells prior to passaging can compromise their transfection efficiency e On the day before transfection plate cells such that they will be at the appropriate confluence at the time of transfection see manufacturer s recommendations for the transfection reagent you are using Example If you are using Lipofectamine 2000 as a transfection reagent plate cells such that they will be 90 95 confluent at the time of transfection e Transfect your plasmid construct into the 293FT Cell Line using the method of choice see above e After transfection add fresh growth medium containing 500 ug ml Geneticin and allow the cells to recover for 24 48 hours before proceeding to assay for expression of your gene of interest 293FT cells can be used as hosts to generate a stable cell line expressing your gene of interest from most plasmids see Note below Remember that the introduced plasmid must contain a selection marker other than neomycin resistance Stable cell lines can then be generated by transfection and dual selection with Geneticin and the appropriate selection agent Since 293FT cells stably express the SV40 large T antigen we do not recommend generating stable cell lines with plasmids that contain the SV40 origin of replication Appendix Map of pCMVSPORT6TAg
12. nes 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 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 res
13. roducts beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 10 Purchaser Notification Introduction Information for European Customers Limited Use Label License No 5 Invitrogen Technology Use of the 293FT Cell Line is covered under the licenses detailed below The 293FT Cell Line is genetically modified and carries the pUC derived plasmid pCMVSPORT6TAg neo As a condition of sale use of this product must be in accordance with all applicable local legislation and guideli
14. t for generating lentiviral constructs using the ViraPower Lentiviral Expression System available from Invitrogen Catalog nos K4950 00 and K4960 00 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 293 F Cell Line available from Invitrogen Catalog no 11625 is a fast growing variant of the 293 cell line and was originally obtained from Robert Horlick at Pharmacopeia 293FT cells stably express the neomycin resistance gene from pCMVSPORT6TAg neo and should be maintained in medium containing Geneticin at the concentration listed below Expression of the neomycin resistance gene in 293FT cells is controlled by the SV40 enhancer promoter Methods General Information General Cell Handling Before Starting Follow the general guidelines below to grow and maintain 293FT cells Make sure that all solutions and equipment that come in contact with the cells are sterile Always use proper sterile technique and work in a laminar flow hood Before 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 For gener
15. t of cell suspension and amount of trypan blue divided by the amount of cell suspension Incubate the cells with the trypan blue solution for 1 2 minutes Count all cells including the blue cells using a Coulter Counter or manually using a hemocytometer chamber To calculate the total cells per ml in suspension multiply the total count by the dilution factor To determine the viability count only the blue cells Calculate the viability 1 00 Number of blue cells Number of total cells x 100 8 Cell viability should be at least 95 for healthy log phase cultures Continued on next page Subculturing Cells continued Subculturing Cells Use this procedure to subculture 293FT cells grown in a T 75 cm flask If you are using other sized flasks scale the reagent volumes accordingly 1 Remove all medium from the flask and wash the cells once with 10 ml PBS to remove excess medium and serum Serum contains inhibitors of trypsin Add 2 ml of trypsin versene EDTA solution to the monolayer and incubate 1 5 minutes at room temperature until cells detach Check the cells under a microscope and confirm that most of the cells have detached If cells are still attached incubate a little longer until most of the cells have detached Add 8 ml complete medium containing Geneticin and transfer the cell suspension to a 15 ml sterile conical tube Determine viable and total cell counts see procedure above Seed cells
16. tamine 2000 Reagent 0 75 ml 11668 027 1 5 ml 11668 019 Dulbecco s Modified Eagle Medium 500 ml 11965 092 DMEM 1000 ml 11965 084 Fetal Bovine Serum 100 ml 16000 036 500 ml 16000 044 10 mM MEM Non Essential Amino Acids 100 ml 11140 050 Solution 200 mM L Glutamine 100 ml 25030 081 MEM Sodium Pyruvate Solution 100X 100 ml 11360 070 Penicillin Streptomycin 100 ml 15070 063 Trypsin EDTA 100 ml 25300 054 Geneticin Selective Antibiotic 1g 11811 023 5g 11811 031 20 ml 50 mg ml 10131 035 100 ml 50 mg ml 10131 027 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 31985 070 Phosphate Buffered Saline PBS pH 7 4 500 ml 10010 023 1L 10010 031 Trypan Blue Stain 100 ml 15250 061 Introduction Product Information 293FT Cell Line Use of the Cell Line Parental Cell Lines Antibiotic Resistance The 293FT Cell Line is a very suitable host for lentiviral production The 293FT Cell Line is derived from the 293F Cell Line see below and stably expresses the SV40 large T antigen from the pCMVSPORT6TAg neo plasmid Expression of the SV40 large T antigen is controlled by the human cytomegalo virus CMV promoter and is high level and constitutive For more information about pCMVSPORT6TAg neo see the Appendix page 9 Studies have demonstrated maximal virus production in human 293 cells expressing SV40 large T antigen Naldini et al 1996 making the 293FT Cell Line a particularly suitable hos
17. ted Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 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 Naldini L Blomer U Gage F H Trono D and Verma I M 1996 Efficient Transfer Integration and Sustained Long Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector Proc Natl Acad Sci USA 93 11382 11388 Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Southern P J and Berg P 1982 Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under Control of the SV40 Early Region Promoter J Molec Appl Gen 1 327 339 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trad
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