BG01V/hOG Cells - Thermo Fisher Scientific
Contents
1. BGO1V Genome Engineered cells will express EmGFP under the appropriate conditions when the Oct4 promoter is active This promoter is only active when hESCs are in the pluripotent state and therefore acts as a sensitive indicator of differentiation Pan et al 2002 This tool can be used as a reporter of the cell culture s response to external stimuli including media composition and stress Methods General Information General Cell Follow the general guidelines below to grow and maintain Handling BGO1V hOG Cells 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 BGO1V hOG Cells may be cultured on a feeder layer of mitotically inactivated mouse embryonic fibroblasts MEFs or may be thawed on MEFs and then transitioned to MEF conditioned media MEF CM or STEMPRO hESC SFM Make sure to start preparing the feeder layer two days before thawing BG01V hOG Cells Before starting experiments be sure to have cells established at least 5 passages and also have some frozen stocks on hand We recommend using early passage cells for your experiments below 30 passages Upon receipt of the cells from Invitrogen grow and freeze multiple vials of the BGO1V hOG cells to ensure that you have an adequate supply of early passage cells For general maintenance of cells pass BG01V hOG Cells before colonies start contacting each othe2. 328 Phi C31 Recombinase Technology This product and its use is the subject of one or more of U S Patent Nos 5 491 084 and 6 146 826 and foreign equivalents This product is sold under license from Columbia University Rights to use this product are limited to research use only and expressly exclude the right to manufacture use sell or lease this product for use for measuring the level of toxicity for chemical agents and environmental samples in cells and transgenic animals No other rights are conveyed Not for human use or use in diagnostic or therapeutic procedures Inquiry into the availability of a license to broader rights or the use of this product for commercial purposes should be directed to Columbia Innovation Enterprise Columbia University Engineering Terrace Suite 363 New York New York 10027 This product and its use is the subject of one or more of U S Patent Nos 5 777 079 6 066 476 and 6 319 669 and foreign equivalents Any use of this product by a commercial entity requires a separate license from either GE Healthcare or Invitrogen Corporation For information on obtaining a commercial license to use this product please refer to the contact information located at the bottom of this statement No rights are conveyed to modify or clone the gene encoding GFP contained in this product For information on licensing contact Licensing Department Invitrogen Corporation 5791 Van Allen Way Carlsbad California 9200
3. a final concentration of 4ng ml before using Preparing Thaw STEMPRO hESC Supplement in 37 C water bath Complete minimize dwell time and prepare according to the table STEMPRO below hESC SFM Component Final conc For 500 ml For 100 ml DMEM F 12 with GLUTAMAX 1X 1X 454 ml 90 8 ml STEMPRO hESC SFM Growth 1X 10 ml 2ml Supplement 50X 25 BSA 1 896 36 ml 7 2ml FGF basic 10 ug ml 8ng ml 400 pl 80 ul 2 Mercaptoethanol 55 mM 0 1 mM 909 ul 182 pl Storing Store complete STEMPRO hESC SFM at 2 8 C in the dark for Complete up to 7 days Add 2 Mercaptoethanol daily during storage Medium at volumes listed in the table above Preparing For the wash medium prepare 0 1 BSA in DMEM F 12 Wash Medium with GLUTAMAX use the 25 BSA provided in the kit BGO1V hOG cells should be cultured in the presence of Important 50 ug ml Hygromycin B to prevent losing the GFP expression cassette during prolonged culturing Continued on next page 13 Culturing on STEMPRO hESC SFM Continued Transferring After thawing and establishing BGO1V hOG cells on MEF Cells into feeders as described on pages 6 7 use the following STEMPRO procedure to transfer the cells into STEMPRO hESC SFM hESC SFM 1 Aspirate medium from the cells and add 1 ml of 1 mg ml collagenase solution for every 10 cm of culture vessel surface area 2 Incubate in a 37 C incubator until the edge of colonies curl up usually it is less than an hour
4. at 37 C humidified atmosphere of 5 CO in air Prepare 10 ug ml FGF basic in DMEM F 12 with 0 1 BSA Aliquot 80 ul per tube and freeze at 20 C Thaw the Geltrex bottle at 2 8 C overnight and prepare 1 ml aliquots of Geltrex in 50 ml conical tubes Store the tubes at 20 C 1 Thaw a 1 ml tube of Geltrex at 2 8 C 2 Remove DMEM F 12 from 2 8 C storage and add 29 ml of cold DMEM F 12 to the 1 ml of Geltrex Mix gently 3 Cover the whole surface of each culture plate with the Geltrex solution 1 ml for a 35 mm dish 1 5 ml fora 60 mm dish 4 Sealeach dish with parafilm to prevent drying and incubate 1 hour at 37 C 5 Transfer each dish to a laminar flow hood and allow it to equilibrate to room temperature about 1 hour before using The Geltrex coated dish may be stored at 2 8 C for up to 1 week Continued on next page Culturing on STEMPRO hESC SFM Continued Preparing Collagenase IV Prepare 1 mg ml and 10 mg ml aliquots of collagenase IV in DMEM F 12 Filter to sterilize and freeze at 20 C Preparing 1 Prepare mitotically inactive MEF cells as described on MEF pages 4 5 Conditioned 2 Change the MEF medium to hESC medium prepared Medium on as page 5 after a 24 hour incubation MEF CM 3 Collect medium from the cells every 24 hours and supplement with FGF basic prepared as described on the previous page at
5. in isopropanol chamber Transfer frozen vials to liquid nitrogen tank for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen 17 Freezing Cells Cultured on SFM Introduction When freezing BGO1V hOG Cells cultured on STEMPRO hESC SFM we recommend the following e Freeze cells at a density of 2 5 x 10 viable cells ml e For every 20 cm of cells one 60 mm dish prepare 0 5 ml of SFM Freezing Medium 1 and 0 5 ml of SFM Freezing Medium 2 e Bring BG01V hOG Cells into freezing medium in two steps as described in this section Guidelines for preparing freezing medium and freezing cells are provided in this section Materials You will need to have the following reagents on hand before Needed beginning see page v for ordering information e Plates with BGO1V hOG Cells in SFM Wash Medium see page 13 e DMEM F 12 with GLUTAMAX e Collagenase Type IV working solution 10 mg ml in DMEM F12 e Knockout Serum Replacement KSR e DMSO use a bottle set aside for cell culture open only in a laminar flow hood Disposable sterile 15 ml conical tubes e Sterile freezing vials Preparing SFM Prepare SFM Freezing Medium 1 and 2 immediately before Freezing use Discard any unused medium Medium 1 Ina sterile 15 ml tube mix together the following for every 0 5 ml of SFM Freezing Medium 1 needed DMEM F12 with GLUTAMA
6. to see this curling 3 Aspirate collagenase solution and add MEF CM supplemented with FGF basic as described on page 13 4 Gently scrape the dish using a 5 ml serological pipette and transfer clumps into a 15 ml tube Try not to make the clumps too small there should be gt 100 cells per clumps 5 Remove the Geltrex from a Geltrex coated plate by tipping the plate slightly and aspirating the solution Wash the plate once with DMEM F12 Take care to avoid drying of the plate surface before plating Seed the cells onto the plate 7 Add a final concentration of 50 ug ml Hygromycin B 1 1 000 dilution of 50 mg ml Hygromycin B stock 8 Place the plate with the cells in the incubator Shake the plate gently to evenly distribute the cells 9 Thenext day feed the cells once more with MEF CM supplemented with FGF basic 10 The following day Day 3 feed the cells with an equivalent amount of complete STEMPRO hESC SFM and a final concentration of 50 pg ml Hygromycin B 11 Thereafter continue feeding cells daily with complete SFM and 50 ug ml Hygromycin B Continued on next page 14 Culturing on STEMPRO hESC SFM Continued Passaging 1 Cells 10 11 12 13 14 15 16 TZ In a 37 C water bath warm appropriate amounts of 10 mg ml collagenase solution complete STEMPRO hESC SFM and wash medium prepared as described on page 13 Minimize dwell time Set up the plate with BGO
7. 1V hOG cells on a dissecting microscope in a biosafety cabinet or laminar flow to comfortably observe colonies Cut out and remove any overtly differentiated colonies with a 21 gauge needle Aspirate the medium and gently add 1 2 ml of collagenase solution Alternatively use the StemPro EZPassage Disposable Stem Cell Passaging Tool to cut the cell colonies into pieces follow the protocol provided with the tool and then proceed to step 7 Leave for 3 minutes to dislodge cells from the substrate Remove collagenase and rinse with DPBS Add 3 ml of wash medium 0 196 BSA in DMEM F 12 with GLUTAMAX see page 13 Gently scrape the dish using a sterile 1000 ul pipette tip Gently transfer the cell clumps using a 5 ml pipette and place into a 15 ml tube Wash plate with 3 ml of wash medium and add to the tube Spin cells at 200 x g for 2 minutes at room temperature Gently aspirate the media and flick the tube to loosen cells Gently resuspend the cells in warm complete STEMPRO hESC SFM using a 1 ml or 5 ml serological pipette Remove the Geltrex from a Geltrex coated plate by tipping the plate slightly and aspirating the solution Immediately plate the cells Do not allow the surface to dry out before plating Mix plates gently to evenly spread out clumps and place the plate in a 37 C incubator at with 5 CO in air Each day gently change the media to remove excess cells and provide fresh nutrients Observe cells eve
8. 3 Spin cells down for 4 minutes at 200 x g 4 Aspirate supernatant 5 Resuspend cells in hESC Medium 2 ml for a 35 mm dish 6 Aspirate feeder layer plates and plate resuspended BGO1V hOG Cells on the prepared MEFs 7 Grow cells in a 37 C incubator with a humidified atmosphere of 5 CO Change the medium every day Following thawing you can continue to culture cells on MEF feeders as described starting on page 8 or you can transition the cells into StemPro hESC SFM as described starting on page 10 Continued on next page Thawing and Establishing Cells Continued Judging Observe colonies recovered at day 5 after thawing to assess Thawed Cells growth rate and differentiation state keeping the following in mind e Cells should be undifferentiated They should express EmGFP if you are unfamiliar with fluorescence microscopy see page 25 and grow as colonies see image below for examples of undifferentiated BG01V hOG Cells If not refer to the Troubleshooting section page 22 e Before colonies start contacting each other they should be passed pee next page TAa BGO1V hOG Cells Culturing on MEF Feeders Introduction Important Materials Needed Important Collagenase Preparation Follow the protocol below to culture BGO1V hOG Cells on feeder layer plates For culturing cells in serum free medium without feeders see page 10 Before starting experiments we recommend t
9. 3 Fountain Drive Tel 1 760 603 7200 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 E mail E mail tech supportQinvitrogen com jpinfoGinvitrogen com eurotech invitrogen com Material Safety Data Sheets MSDSs Certificate of Analysis 26 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Purchaser Notification Information for European Customers Limited Warranty BGO1V hOG Cells variant hESC hOct4 GFP Reporter Cells are genetically modified and carry a GFP reporter and a Hygromycin Resistance gene The paternal human embryonic stem cells were derived March 2001 from a supernumerary IVF embryo that would have otherwise been discarded and was obtained with informed consent 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 Invitrogen is committed to providing our customers with high quality goo
10. 8 Phone 760 603 7200 Email outlicensing invitrogen com This product and its use is the subject of one or more of U S Patent Nos 6 090 919 5 804 387 5 994 077 and foreign equivalents This product and its use are the subject of one or more of U S Patent Nos 6 632 672 and 7 361 641 and foreign equivalents 29 References Mitalipova M Calhoun J Shin S Wininger D Schulz T Noggle S Venable A Lyons I Robins A and Stice S 2003 Human embryonic stem cell lines derived from discarded embryos Stem Cells 21 521 526 Pan G J Chang Z Y Scholer H R and Pei D 2002 Stem cell pluripotency and transcription factor Oct4 Cell Res 12 321 329 Plaia T W Josephson R Liu Y Zeng X Ording C Toumadje A Brimble S N Sherrer E S Uhl E W Freed W J Schulz T C Maitra A Rao M S and Auerbach J M 2006 Characterization of a new NIH registered variant human embryonic stem cell line BGO1V a tool for human embryonic stem cell research Stem Cells 24 531 546 2007 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 30 Notes Notes invitrogen Corporate Headquarters Invitrogen Corporation 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 v
11. EM F12 with GLUTAMAX 2 mM Knockout Serum Replacement KSR e bFGF Reconstitute lyophilized human bFGF in sterile DMEM F12 containing 0 1 BSA to 10 ug ml Divide stock solution into working aliquots and store at lt 20 C Porcine skin gelatin Prepare 0 1 w v porcine skin gelatin Sigma Cat no G1890 in sterile distilled water and sterilize by filtration using a 0 2 um filter Store up to 1 year at 4 C e 37 C incubator with a humidified atmosphere of 5 CO Continued on next page Preparing a Feeder Cell Layer Continued Preparing MEF To prepare 500 ml MEF medium mix the following reagents Medium Volume Reagent Final Concentration 445 ml DMEM 1x 50 ml FBS 10 5ml NEAA 10 mM 0 1 mM 500 pl 2 Mercaptoethanol 55 uM 1 000X 55 mM Filter through 0 22 uM filtration unit Pre heat the medium to 37 C before use Preparing To prepare 100 ml hESC Medium mix following reagents hESC Medium Volume Reagent Final Concentration 79 ml DMEM F12 with 1x GLUTAMAX 20 ml Knockout Serum 20 Replacement KSR 1ml NEAA 10 mM 0 1 mM 100 ul 2 Mercaptoethanol 55 uM 1 000X 55 mM 40 ul bFGF 10 pg ml 4ng ml If stored at 4 C hESC Medium can be kept for up to 1 week Pre heat the medium to 37 C before use Preparing Coat plates for 20 60 minutes at room temperature with Gelatin Coated 0 1 gelatin in dH20 Plates Plating Feeder 1 Two days before hESC coculture plate 30 000 cm of Layer mitot
12. Fs DR4 strain 1 Culture MEFs in MEF medium see page 5 2 Inactivate by treating MEFs with 10 pg ml mitomycin C for 2 to 3 hours at 37 C 3 Wash cells four times with Dulbecco s Phosphate Buffered Saline D PBS Cat no 14190 144 4 Trypsinize cells with 0 05 Trypsin EDTA Cat no 25300 054 5 Plate MEFs at a density of 3 x 10 cells cm of culture surface area in MEF medium see page 5 with 2 5 ml per well of a gelatin coated 6 well dish 6 Freeze the cells for later use or use within 2 to 5 days after plating for hESC cell culture The medium should be changed every other day if they are not used immediately Detecting Fluorescence Introduction Filters for Use with EmGFP Fluorescence Microscope Color Camera What You Should See You may detect EmGFP protein expression directly in undifferentiated BGO1V hOG Cells by fluorescence microscopy or other methods that use light excitation and detection of emission See below for recommended fluorescence microscopy filter sets The EmGFP can be detected with standard FITC filter sets However for optimal detection of the fluorescence signal you may use a filter set which is optimized for detection within the excitation and emission ranges for the fluorescent protein such as the Omega XF100 filter set for fluorescence microscopy The spectral characteristics of EmGFP are listed in the table below Fluorescent Protein Excitation nm Emission n
13. RC 1045 2 Hygromycin resistant MEFs DR4 are also available separately from ATCC Cat no SCRC 1045 Mitomycin C is available separately from Sigma St Louis Cat no M0503 Continued on next page Accessory Products Continued Fetal Bovine Invitrogen also provides ES Cell Qualified Fetal Bovine Serum ES Serum originating from countries other than the U S go to Cell Qualified www invitrogen com for information These can be more appropriate for your situation and may be used to grow BG01V hOG Cells Porcine Skin Porcine Skin Gelatin can be obtained from Sigma St Louis Gelatin Cat no G1890 vi Introduction Introduction Characteristics of BG01V hOG Cells BGO1V Parental Cell Line BG01V hOG human embryonic stem cells RESCs are engineered to enable monitoring the pluripotency of hESCs without sacrificing cells When pluripotent these cells express Emerald Green Fluorescent Protein emGFP Use these cells to assess whether your culture conditions are adequate to keep hESCs undifferentiated or determine the efficiency of your differentiation assays BG01V hOG cells have the following characteristics Prepared from low passage passage 17 parental BGO1V cells cultured on murine embryonic feeders MEFs e Pluripotent can differentiate to representatives of the three primary germ layers e Express EmGFP when pluripotent lose EmGFP expression upon differentiation BG01
14. V hOG Cells are derived from the BGO1V hESC line ATCC No SCRC 2002 BGO1V cells in turn are a variant with abnormal karyotype of the wild type parental hESC line BGO1 Mitalipova et al 2003 Plaia et al 2006 BGO1V cell colonies grow on murine embryonic feeders MEFs with uniform morphology and are easy to culture at a predictable growth rate BGO1V cells stain positive for pluripotency markers and alkaline phosphatase activity BGO1V cells are pluripotent and can differentiate to representatives of all three primary germ layers Continued on next page Introduction Continued Generation of BG01V hOG Cells GFP Expression Indicates Pluripotency We constructed an integration vector containing EmGFP expressed under the direction of the human Oct4 promoter and followed by the HSV TK polyadenylation signal This plasmid was stably integrated into the genome of BGO1V cells We selected a clone expressing EmGFP which was tested extensively to make sure it was pluripotent and able to differentiate to representatives of the three primary germ layers The resulting cell line was called BG01V hOG Note Since the integration vector used contains a Hygromycin resistance gene Hyg BG01V hOG Cells are resistant to Hygromycin B If you want to stably integrate more genes in these cells do not use the Hygromycin resistant gene as your selection marker hOct EmGFPpA i hOct4 GFP c gt Reporter MM Construct Hyg
15. X 0 5 ml Knockout Serum Replacement KSR 0 5 ml 2 In another sterile 15 ml tube mix together the following for every 0 5 ml of SFM Freezing Medium 2 needed DMEM F12 with GLUTAMAX 0 8 ml DMSO 0 2 ml 3 Place tube with SFM Freezing Medium 2 on ice and leave SFM Freezing Medium 1 at room temperature Continued on next page 18 Freezing Cells Cultured on SFM Continued Freezing Cells 1 Cultured on SrEMPRO hESC SFM 10 11 12 Aspirate serum free culture medium from the cells and gently add 1 2 ml of 10 mg ml collagenase solution Alternatively use the StemPro EZPassage Disposable Stem Cell Passaging Tool to cut the cell colonies into pieces follow the protocol provided with the tool and then proceed to step 4 Leave for 3 minutes to dislodge cell colonies from the substrate Remove collagenase and rinse with DPBS Add 3 ml of wash medium 0 1 BSA in DMEM F 12 with GLUTAMAX see page 13 Gently scrape the dish using a sterile 1000 l pipette tip Gently transfer the cell clumps using a 5 ml pipette and place into a 15 ml tube Wash plate with 3 ml of wash medium and add to the tube Spin cells down for 2 minutes at 200 x g at room temperature Gently aspirate media and resuspend the BGO1V hOG cells in SFM Freezing Medium 1 at room temperature use 0 5 ml of Freezing Medium 1 for one 60 mm dish Add the same volume of cold SFM Freezing Medium 2 to cells in a dropwise manner swirling t
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17. ew batch of mitotically inactivated MEFs if necessary Use Hygromycin resistant MEFs Culture conditions Thaw and culture fresh vial of BGO1V hOG Cells not correct Follow thawing instructions page 6 and subculture procedures page 8 exactly No Incorrect filters used Be sure to use the recommended filter sets for fluorescence to detect detection of fluorescence see page 25 Be sure to signal detected fluorescence use an inverted fluorescence microscope for analysis Cells lost GFP Thaw and culture fresh vial of BGO1V hOG Cells expression cassette Culture cells in presence of 50 ug ml Hygromycin B Cells differentiated See points above 23 Appendix Generating Mitomycin C Treated MEFs Preparing Gelatin Coated Plates Preparing Mitomycin C Obtaining MEFs Mitomycin C Treatment 24 Mitomycin C is highly toxic Read and understand the MSDS and handle accordingly Prepare 0 1 w v porcine skin gelatin Sigma Cat no G1890 in sterile distilled water and sterilize by filtration using a 0 2 um filter Store up to 1 year at 4 C Coat plates for 20 to 60 minutes at room temperature with 0 1 gelatin in dH20 Prepare 10 ug ml mitomycin C in MEF medium filter sterilize and store at 20 C until use Good for 2 weeks at 4 C Obtain Hygromycin resistant MEFs DR4 from ATCC Cat no SCRC 1045 Use the procedure below to generate mitotically inactivated ME
18. hat you first prepare ample cell stocks as described in Freezing Cells page 16 You will need to have the following reagents on hand before beginning see page v for ordering information e Plates with BGO1V hOG Cells e hESC Medium see page 5 for composition pre warm to 37 C before use e DMEM F12 with GLUTAMAX e Feeder layer plates with mitotically inactivated MEFs prepare at least two days in advance see page 4 e Collagenase Type IV e Hygromycin B e Disposable sterile 15 ml tubes e 37 C incubator with humidified atmosphere of 5 CO BG01V hOG Cells should be cultured in the presence of 50 ug ml Hygromycin B on order to prevent losing the GFP expression cassette during prolonged culturing Prepare 1 mg ml aliquots of collagenase IV in DMEM F 12 Filter to sterilize and freeze at 20 C Continued on next page Culturing on MEF Feeders Continued Passaging Cells 1 10 Aspirate culture medium and add 1 ml of 1 mg ml collagenase solution for every 10 cm of culture vessel surface area Alternatively use the StemPro EZPassage Disposable Stem Cell Passaging Tool to cut the cell colonies into pieces follow the protocol provided with the tool and then proceed to step 4 Incubate in a 37 C incubator until the edge of colonies curl up usually less than an hour Aspirate collagenase solution Add hESC Medium or 0 1 BSA in DMEM F12 Gently scrape dish using a 5 ml
19. he tube after each drop Resuspend the cells by gently pipetting 2 3 times Aliquot 1 ml of the cell suspension to each freezing vial and store at 80 C overnight in isopropanol chamber Transfer frozen vials to liquid nitrogen tank for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen 19 Expected Results Introduction Pluripotent BGO1V hOG Cells express EmGFP but upon differentiation these cells lose EmGFP expression In this section some typical examples are shown to help you interpret your experiments Note Suggestions for differentiation protocols are available from www invitrogen com stemcells click on the section protocols FACS Analysis BG01V hOG human embryonic stem cells differentiate when induced to form embryoid bodies Embryoid body derived BG01V hOG Cells hardly contain any EmGFP positive cells as determined by FACS analysis Undifferentiated BGO01V hOG Cells 89 1 GFP positive 18 1 10e1 PM 1 Green Fluorescence mean fluorescence Embryoid Body Derived BG01V hOG Cells 1 1 3 4 GFP positive mean fluorescence 10e0 10e1 10e2 10e3 10e4 PM 1 Green Fluorescence 10e2 10e3 10e4 20 Continued on next page Expected Results Continued Fluorescence EmGFP expression is lost upon embryoid body induced Microscopy differentiation of BGO1V hOG Cells as seen in the green f
20. ically inactivated mouse embryonic fibroblasts on a 0 176 gelatin coated culture plate in MEF medium 2 One day before hESC coculture replace medium with hESC Medium 3 Next day the feeder layer is ready to be seeded with BG01V hOG Cells in fresh hESC Medium Thawing and Establishing Cells Introduction BG01V hOG cells are supplied in a vial containing 1 ml of cells at 2 x 106 viable cells ml in freezing medium They are frozen from cultures grown on MEF feeder cells and must be thawed on MEF feeders Follow the protocol below to thaw BGO1V hOG Cells directly into hESC Medium in a 35 mm dish Materials You will need to have the following reagents on hand before Needed beginning see page v for ordering information e BGOIV hOG cells store frozen cells in liquid nitrogen until ready to use e hESC Medium see page 5 for composition pre warm to 37 C before use e Feeder layer plates with mitotically inactivated MEFs prepare at least two days in advance see page 4 e Disposable sterile 50 ml tubes e 37 C incubator with humidified atmosphere of 5 CO Thawing Store frozen cells in liquid nitrogen until ready to use To Procedure thaw and establish BG01V hOG Cells 1 Remove the cryovial of cells from the liquid nitrogen and thaw quickly in a 37 C water bath to prevent crystal formation 2 When thawed immediately transfer cells into 50 ml tube and add warm hESC Medium dropwise up to 10 ml
21. invitrogen BGO01V hOG Cells Variant hESC hOct4 GFP Reporter Cells Catalog no R7799 105 A10022 Version D 30 September 2008 Table of Contents Contents and Storage eee etse eet eet ete iv Accessory Products eet rece atn metre ia v Introduction i pet rt ep ROEA 1 Methods TT 3 General Information iiia ieie entier i eee ie abe add 3 Preparing Feeder Cell Layer oot dete eerte Thawing and Establishing Cells Culturing on MEE Feeders eee tenerent 8 Culturing on STEMPRO hESC SEM sert 1t ener 10 Freezing Cells Cultured on MEF Feeders sese 16 Freezing Cells Cultured on SFM sss 18 Expected Res lts unatesaisueninuen esee eaae ned n 20 Troubleshooting ener ener 22 Appendix cerne triti itm e a aaas ae e iere ier ii ee antt erste 24 Generating Mitomycin C Treated MEFS sss 24 D tecting Fluorescence omine sabe nae nre da 25 Technical Support eee eene d E 26 Purchaser Notificatio oc rct c eret 27 R f rences teet te R b e EX EE i dere eee HT 30 Contents and Storage Shipping and Storage Contents o This manual is shipped with BGO1V hOG Cells BG01V hOG cells are shipped on dry ice Upon receipt store in liquid nitrogen Storage conditions Liquid nitrogen Amount supplied One vial containing 2 x 10 cells Composition 1 ml of cells in Freezing medium see page 10 f
22. isit our web site at www invitrogen com
23. luorescence image below Undifferentiated Embryoid Body Derived BGO01V hOG Cells BGO1V hOG Cells Brightfield image HORSE E Green fluorescence image Note If you are unfamiliar with fluorescence microscopy see page 25 21 Troubleshooting Culturing The table below lists some potential problems and solutions that Cells help you troubleshoot your cell culture problems Problem Cause Solution No viable Stock not stored Order new stock and store in liquid nitrogen cells after correctly Keep in liquid nitrogen until thawing thawing Home made stock not Freeze cells at a density of 2 5 x 10 viable stock viable cells ml Use low passage cells to make your own stocks Follow the freezing procedure for your type of cell culture starting on page 16 exactly Slow freezing and fast thawing are key Add the cold freezing medium in a dropwise manner slowly swirling the tube after each drop At the time of thawing thaw quickly and do not expose vial to the air but quickly change from nitrogen tank to 37 C water bath Obtain new BGO1V hOG Cells Thawing medium not Use specified medium correct Cells too diluted Generally we recommend thawing one vial in a 35 mm dish If you need to concentrate cells spin down the culture for 4 minutes at 200 x g at room temperature and dilute the cells at higher density MEFs sub optimal and Purchase see page v or make see page 24 a new do
24. m EmGFP 487 509 For information on obtaining these filter sets contact Omega Optical Inc www omegafilters com or Chroma Technology Corporation www chroma com You may view the fluorescence signal of EMGFP in cells using an inverted fluorescence microscope with FITC filter or Omega XF100 filter available from www omegafilters com for viewing cells in culture or a flow cytometry system If desired you may use a color camera that is compatible with the microscope to photograph the cells We recommend using a digital camera or high sensitivity film such as 400 ASA or greater See the Expected Results Section page 21 25 Technical Support Web Resources Visit the Invitrogen Web site 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 Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan Minato ku
25. not support batch of mitotically inactivated MEFs recovery of BG01V hOG Cells MEFs MEFs not inactivated Inactivate mitosis in MEFs as described on page overgrow 24 or purchase inactivated MEFs see page v plate 22 Continued on next page Troubleshooting Continued Culturing The table below lists some potential problems and solutions that Cells help you troubleshoot your cell culture problems Problem Cause Solution Cells grow Growth medium Use correct growth medium slowly not correct bFGF inactive bFGF is not stable when frequently warmed and cooled Add bFGF to medium just before use or store medium with bFGF in aliquots at 20 C Cells too old Use healthy BG01V hOG cells under passage 30 do not overgrow Cells too diluted Spin down cells for 4 minutes 200 x g at room temperature aspirate media and dilute cells at higher density Clump size is to Be gentle at time of passage so the clumps of cells small and don t get too small differentiated Mycoplasma Discard cells media and reagents and use early contamination stock of cells with fresh media and reagents Cells Cells not thawed Thaw and culture a fresh vial of BGO1V hOG cells differentiated and established on Make sure to thaw on feeder layers as described on feeder layers page 4 Suboptimal quality Check the concentration of feeder cells used of feeder layer Purchase see page v or make see page 24 n
26. or composition Handle as potentially biohazardous material under at least Biosafety Level 1 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet before handling Accessory Products Additional For more information about the following products refer to Products our Web site www invitrogen com or call Technical Support see page 24 Item Quantity Catalog no Collagenase Type IV 1g 17104 019 DMEM F12 containing GLUTAMAX 2mM 500 ml 10565 018 Knockout Serum Replacement KSR 500 ml 10828 028 MEM Non Essential Amino Acids Solution 10 mM 100 ml 11140 050 100X FGF Basic bFGF 50 ug PHG0026 STEMPRO hESC SFM 1 kit A1000701 2 Mercaptoethanol 1 000X 55 mM 50 ml 21985 Dulbecco s Modified Eagle Medium D MEM high 500 ml 11995 065 glucose with L glutamine and sodium pyruvate Fetal Bovine Serum ES Cell Qualified US 500 ml 16141 079 Bovine Albumin Fraction V Solution 7 5 100 ml 15260 037 Geltrex 5 ml 12760 DPBS 500 ml 14190 Antibiotic Antimycotic 100X liquid 100 ml 15240 062 Hygromycin B 20 ml 10687 010 StemPro EZPassage Disposable Stem Cell 10 disposable 23181 010 Passaging Tool tools StemPro EZChek Human Tri Lineage Multiplex 100 reactions 23191 050 PCR Kit Water distilled 500 ml 15230 162 Mitomycin C Mitomycin C treated Hygromycin resistant MEFs DR4 are Treated MEFs available from ATCC Cat no SC
27. r When thawing or subculturing cells transfer cells into pre warmed medium 10 ml L of Antibiotic Antimycotic containing penicillin streptomycin and amphotericin B may be used if required see page v for ordering information Itis very important to strictly follow the guidelines for culturing BGO1V hOG Cells in this manual to keep them Important P undifferentiated As with other human cell lines when working with BGO01V hOG cells handle as potentially biohazardous material under at least Biosafety Level 1 containment Preparing a Feeder Cell Layer Introduction BGO1V hOG cells are frozen from cultures grown on mouse embryonic fibroblast MEF feeder cells and should be thawed on feeders Follow the protocol below to prepare the feeder layer matrix Use mitotically inactivated MEFs to prevent overgrowth of the hESCs Both Mitomycin C and irradiation methods can be used to mitotically inactivate your MEFs Materials Have the following reagents on hand before beginning see Needed page v for ordering information Mitotically inactivated Hygromycin resistant MEFs Order from ATCC SCRC 1045 2 or generate them as described in Generating Mitomycin C Treated MEFs page 24 e Dulbecco s Modified Eagle Medium D MEM high glucose with L glutamine and sodium pyruvate e Fetal Bovine Serum ES Cell Qualified e MEM Non Essential Amino Acids Solution 10 mM 100X NEAA e 2 Mercaptoethanol 1 000X e DM
28. r warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Continued on next page 27 Purchaser Notification Continued Limited Use Label License 5 Invitrogen Technology 28 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 componen
29. ry 1 ml of Freezing Medium B needed hESC Medium 0 8 ml DMSO 0 2 ml Place tube with Freezing Medium B on ice and leave Freezing Medium A at room temperature Continued on next page Freezing Cells Cultured on MEF Feeders Continued Freezing Cells Cultured on MEF Feeders aA 10 Aspirate culture medium from the cells and add 1 ml of 1 mg ml collagenase solution for every 10 cm of culture vessel surface area Alternatively use the StemPro EZPassage Disposable Stem Cell Passaging Tool to cut the cell colonies into pieces follow the protocol provided with the tool and then proceed to step 4 Incubate in a 37 C incubator until the edge of colonies curl up usually less than an hour Aspirate collagenase solution Add hESC Medium see page 5 or 0 1 BSA in DMEM F12 Gently scrape dish using 5 ml serological pipette and transfer clumps into a 15 ml tube Try not to make the clumps too small there should be 100 cells per clumps see page 9 for an example Spin cells down for 2 minutes at 200 x g at room temperature Gently aspirate media and resuspend BGO1V hOG cells in Freezing Medium A e g resuspend cells from one 60 mm dish in 1 ml of freezing medium Add the same volume of Freezing Medium B to cells in a dropwise manner swirling the tube after each drop Resuspend the cells by gently pipetting 2 3 times Aliquot 1 ml of the cell suspension to each freezing vial and store at 80 C overnight
30. ry day and passage by the above protocol whenever required about every 5 7 days 15 Freezing Cells Cultured on MEF Feeders Introduction Materials Needed Preparing Freezing Medium 16 When freezing BGO1V hOG Cells that are cultured on MEF feeder cells we recommend the following Freeze cells at a density of 2 5 x 10 viable cells ml For every 20 cm of cells one 60 mm dish prepare 1 ml of MEF Freezing Medium A and 1 ml of MEF Freezing Medium B see below Bring BGO1V hOG Cells into freezing medium in two steps as described in this section Guidelines for preparing freezing medium and freezing cells are provided in this section You will need to have the following reagents on hand before beginning see page v for ordering information Plates with BGO1V hOG Cells on MEF feeders hESC Medium see page 5 for composition Collagenase Type IV working solution 1 mg ml in DMEM F12 see page 8 Fetal Bovine Serum ES Cell Qualified DMSO use a bottle set aside for cell culture open only in a laminar flow hood Disposable sterile 15 ml conical tubes Sterile freezing vials Prepare Freezing Medium A and B immediately before use Discard any unused medium 1 In a sterile 15 ml tube mix together the following for every 1 ml of Freezing Medium A needed hESC Medium 0 5 ml Fetal Bovine Serum ES Cell Qualified 0 5 ml In another sterile 15 ml tube mix together the following for eve
31. s we recommend that you first prepare ample cell stocks as described in Freezing Cells page 10 STEMPRO hESC SFM has been extensively tested and proven to have the following characteristics e Supports hESC growth for up to 80 passages maintaining the ability of hESCs to differentiate into all three germ line lineages without any signs of karyotypical abnormalities e Maintains pluripotency in multiple hESC lines including BGO1V cells e Supports scale up production of hESCs to over 1 x 10 cells while maintaining pluripotency e No need to maintain feeder cells or produce feeder conditioned medium e More reproducible results due to steady growth factor levels Continued on next page Culturing on STEMPRO hESC SFM Continued Guidelines for SFM Culture To prevent differentiation and slow growth of BGO1V hOG cells grown in STEMPRO hESC SFM follow these guidelines Starter culture This must be a high quality culture with a high density of cells and primarily undifferentiated The starter culture should be cells maintained on Geltrex in Mouse Embryonic Fibroblast Conditioned Medium MEF CM See the protocol in this section for transferring cells from MEF feeders to MEF CM and then into STEMPRO hESC SFM Passaging It is critical to achieve high plating survival of colony pieces The pieces must be a bit smaller than TM typical collagenase passaging on Geltrex MEF CM Some cell dea
32. serological pipette and transfer clumps into a 15 ml tube Do not make the clumps too small there should be gt 100 cells per clump See below for an example of acceptable clumps Spin cells down for 2 minutes at 200 x g at room temperature Gently aspirate media and resuspend the BG01V hOG Cells in hESC Medium Aspirate feeder layer plates and plate resuspended BG01V hOG Cells on the prepared MEFs passage ratio 1 3 or 1 4 Add a final concentration of 50 ug ml Hygromycin B 1 1 000 dilution of 50 mg ml Hygromycin B stock Grow cells in a 37 C incubator with a humidified atmosphere of 5 CO Change the medium everyday Feed cells every day and passage by the above protocol whenever required before colonies start contacting each other typically every 4 7 days Culturing on STEMPRO hESC SFM Introduction Note Important Features of the Medium 10 STEMPRO hESC SFM allows you to culture BGO1V hOG Cells in a serum free medium SFM without feeder cells Follow the protocol in this section to culture BGO1V hOG Cells using STEMPRO hESC SFM BGO1V hOG Cells are initially frozen down from cells grown on MEF feeders and should be thawed on feeders as described on pages 6 7 before transferring into MEF conditioned medium MEF CM and then into STEMPRO hESC SFM as described in this section Frozen stocks may then be prepared from the cells grown on STEMPRO hESC SFM Before starting experiment
33. th at passaging is normal but wide scale cell death i e lt 20 survival indicates poor passaging Timing of passaging Critical the cultures need to grow to near confluence i e a day or two after the colonies are just touching cultures should be harvested This usually results in a cell density of 2 5 to 4 x 10 cells cm at time of harvest Do not over expose cells to collagenase we recommend 3 minutes at most even with lower amounts of collagenase Density The cultures must be maintained at a high density 200 colonies in a 60 mm dish hESCs grown in culture are always under selection pressure of proliferation vs differentiation The cultures should be fed every day do not exhaust medium by not feeding Scrape clearly differentiated areas out with a 21 gauge needle Continued on next page 11 Culturing on STEMPRO hESC SFM Continued Materials Needed Preparing FGF basic Preparing Geltrex Aliquots Coating Plates with Geltrex 12 You will need to have the following materials on hand before beginning see page v for ordering information e STEMPRO hESC SFM which includes DMEM F 12 with GLUTAMAX STEMPRO hESC Supplement Bovine Serum Albumin 25 BSA e FGF basic 10 pg ml prepared as described below Collagenase Type IV prepared as described on next page e Geltrex e DPBS e 2 Mercaptoethanol Hygromycin B e Culture dishes 60 mm dishes recommended e An incubator
34. ts 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 most restrictive terms apply Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen 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 Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com Continued on next page Purchaser Notification Continued Limited Use Label License 127 GFP with Heterologous Promoter Limited Use Label License 198 Fluorescent Protein Products Limited Use Label License 267 Mutant GFP Products Limited Use Label License
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