USER GUIDE Freedom™ DG44 Kit For transfection of CHO DG44 Cells (cGMP banked) and development of stable cell lines for protein production Catalog Number A13737-01 Publication Number MAN0003649 Revision 3.00 For research use only. Not for use in diagnostic procedures.
For research use only. Not for use in diagnostic procedures. The information in this guide is subject to change without notice. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT.
Contents Product information ........................................................................................................ 5 Kit contents and storage........................................................................................................................................5 Description of the system .....................................................................................................................................
Appendix C: Ordering information ................................................................................ 70 Accessory products .............................................................................................................................................. 70 Appendix D: Safety ........................................................................................................ 72 Chemical safety ......................................................................................
Product information Kit contents and storage Introduction The components of the Freedom™ DG44 Kit and their shipping and storage conditions are listed below.
Description of the system Freedom™ DG44 Kit Components of the Freedom™ DG44 Kit 6 The Freedom™ DG44 Kit is designed for easy cloning and expression of recombinant proteins in dihydrofolate reductase (DHFR)-deficient, Chinese hamster ovary (CHO)-derived CHO DG44 Cells (cGMP banked) in suspension culture.
Description of the system, continued Advantages of the Freedom™ DG44 Kit TOPO® TA Cloning Vector Kits The Freedom™ DG44 Kit provides the following advantages for protein production in mammalian cells: • DHFR-deficient cGMP banked CHO DG44 Cells derived from CHO cells (Urlaub et al., 1983; Urlaub et al., 1986) provide stable and accurate glycosylation (Sheeley et al., 1997; Werner et al., 1998) and are observed to yield accurate glycoproteins.
Experimental flowchart for two-subunit protein expression Introduction The diagram below schematically depicts the steps necessary to express your twosubunit protein of interest using the Freedom™ DG44 Kit, and it shows several common pathways from stable transfectants to clone scale-up. Note that the times shown for various experimental steps are approximations, and the actual times depend on your protein of interest and the specific workflows you choose.
Experimental flowchart for single-subunit protein expression Experimental flowchart for single-subunit protein expression The diagram below schematically depicts the steps necessary to express your single-subunit protein of interest using the Freedom™ DG44 Kit as well as several common pathways from stable transfectants to clone scale-up.
CHO DG44 cells (cGMP banked) Introduction The CHO DG44 cell line is a dihydrofolate reductase (DHFR)-deficient cell line derived from suspension Chinese hamster ovary (CHO) cells (Urlaub et al., 1983; Urlaub et al., 1986). CHO DG44 Cells (cGMP banked), manufactured under cGMP guidelines, are adapted to suspension culture in CD DG44 Medium.
CD DG44 Medium Introduction CD DG44 Medium is a defined, serum-free medium containing hypoxanthine and thymidine for high-density suspension culture of untransfected CHO DG44 Cells. The medium contains no human or animal origin components.
FreeStyle™ MAX Reagent and OptiPRO™ SFM FreeStyle™ MAX Reagent FreeStyle™ MAX Reagent is a proprietary, animal origin-free formulation for the highly efficient transfection of plasmid DNA into eukaryotic cells. FreeStyle™ MAX Reagent is specifically formulated to achieve the highest transfection levels and lowest cytotoxicity in CHO DG44 Cells (cGMP banked) and other suspension cell lines, including FreeStyle™ CHO-S™ and FreeStyle™ 293-F cells.
CD OptiCHO™ Medium Introduction CD OptiCHO™ Medium is a chemically defined, serum-free medium for selection and high-density suspension culture of stably-transfected CHO DG44 Cells (cGMP banked) expressing DHFR and the neomycin resistance gene. If you are expressing a two-subunit protein, you will perform one round of selection on your transfected cells using CD OptiCHO™ medium containing 500 µg/mL of Geneticin® reagent, as detailed on page 31.
Methods Creating expression plasmids for the Freedom™ DG44 Kit Introduction The Freedom™ DG44 Kit contains two vectors, pOptiVEC™-TOPO® and pcDNA™3.3-TOPO. See pages 66–69 for maps and features of each vector. Using the instructions in this manual, you will: • For two subunits proteins, clone two separate PCR products corresponding to each of the two subunits (SU1 and SU2, SU: subunit) of your protein of choice separately into pOptiVEC™-TOPO® TA and pcDNA™3.
Thawing and subculturing CHO DG44 Cells (cGMP banked) Introduction Follow the protocol below to thaw CHO DG44 Cells (cGMP banked). The cells are supplied in a vial that contains 1 mL of cells at 1 × 107 viable cells/mL in 90% complete CD DG44 medium and 10% DMSO. Thaw the cells directly into CD DG44 Medium supplemented with 8 mM L-glutamine and 18 mL of Pluronic® F-68 per liter. IMPORTANT! Do not thaw and grow CHO DG44 Cells (cGMP banked) in CD OptiCHO™ Medium.
Thawing and subculturing CHO DG44 Cells (cGMP banked), continued Thawing procedure 1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly (<1 minute) in a 37°C water bath. 2. Decontaminate the outside of the vial with 70% ethanol. Gently break up any clumps with a sterile pipette tip and aseptically transfer the entire contents of the cryovial into a disposable, sterile polycarbonate 125-mL Erlenmeyer shaker flask containing 29 mL of pre-warmed complete CD DG44 Medium.
Thawing and subculturing CHO DG44 Cells (cGMP banked), continued Subculturing cells Passage the cells every 2–3 days into fresh medium. When passaging CHO DG44 Cells (cGMP banked), use disposable, sterile polycarbonate 125-mL Erlenmeyer shaker flasks with vented caps containing 30 mL of pre-warmed complete CD DG44 Medium (for instructions on preparing complete CD DG44 Medium see page 15). 1. Determine the viable and total cell counts. 2.
Freezing CHO DG44 Cells Introduction You may freeze CHO DG44 Cells (cGMP banked) directly in CD DG44 Medium with 10% DMSO. We recommend that you freeze the cells at a density of ≥1 × 107 viable cells/mL. Guidelines for preparing freezing medium and to freeze cells are provided in this section.
Establishing sensitivity to Geneticin® Selective Antibiotic (G-418) Geneticin® Selective Antibiotic (G-418) The pcDNA™3.3-TOPO TA vector contains the neomycin resistance gene, which confers resistance to the antibiotic Geneticin® Selective Antibiotic (also known as G-418 sulfate). Geneticin® reagent is available separately (see page 70 for ordering information) CAUTION! Geneticin® Selective Antibiotic is harmful. It may cause sensitization by skin contact, and it is irritating to eyes and skin.
Methods for two-subunit protein expression One-page flowcharts Introduction The following pages contain flowcharts to aid you in your expression experiments. Each flowchart consists of an outline of the necessary steps and space for notes that you can then transfer to your laboratory notebook. Using the one-page flowcharts The page numbers by the experimental steps in the flowcharts are hyperlinked to detailed protocols.
Optimizing vectors for two-subunit protein expression Introduction Prior to making stable transfectants in CHO DG44 Cells (cGMP banked), you may perform transient transfections of CHO-S cells or several stable transfections of CHO DG44 Cells (cGMP banked) with various combinations of pOptiVEC™ and pcDNA™3.3 plasmid constructs to determine which vector combination gives optimal protein yield.
Expressing two-subunit proteins without gene amplification Introduction The flowchart below depicts the major steps to express your two-subunit protein in suspension CHO DG44 Cells (cGMP banked) with the combination of pOptiVEC™ and pcDNA™3.3 constructs that gave you the highest yield.
Genomic amplification by MTX addition Introduction The flowchart below depicts the major steps to amplify the copy number of your gene of interest (GOI) using MTX (as methotrexate hydrate) for increased production of the protein of interest.
Performing clonal selection by limiting dilution (two-subunit protein) Introduction The flowchart below depicts the major steps to obtain a clonal cell line (i.e., derived from a single cell) for the production of a two-subunit protein by diluting the pool of stably transfected cells or the MTX-amplified cells to 0.5–2 cells per well in a 96-well plate containing cloning medium. In most cases, one of the cells forms a distinct colony that can later be scaled up.
Transiently transfecting FreeStyle™ CHO-S™ cells Recommendation We recommend FreeStyle™ CHO-S™ cells and transfection with FreeStyle™ MAX Reagent for transient transfection to determine which vector combination gives optimal protein yield. Both FreeStyle™ CHO-S™ cells and FreeStyle™ CHO Expression Medium are available separately, see page 70. Plasmid preparation The pOptiVEC™ and pcDNA™3.
Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for two-subunit protein expression Introduction After determining which combination of pOptiVEC™ and pcDNA™3.3 plasmid constructs gives optimal protein yield using your detection method of choice, you will use FreeStyle™ MAX Reagent to stably transfect suspension CHO DG44 Cells (cGMP banked) with the best combination of vectors.
Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for two-subunit protein expression, continued Optimal transfection conditions To transfect suspension CHO DG44 Cells (cGMP banked) in a 30 mL volume, we recommend using the following optimized conditions: • Final transfection volume: 30 mL • Number of cells to transfect: total of 1.
Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression (optional) Recommendation Calculate the number of CHO DG44 Cells (cGMP banked) that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 95% viable before proceeding to transfection. Materials needed • Suspension CHO DG44 Cells (cGMP banked) cultured in complete CD DG44 Medium • Purified, linearized pOptiVEC™ and pcDNA™3.
Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression, continued Preparing cells 1. 24 hours before transfection, passage CHO DG44 cells at 3 × 105 viable cells/mL in complete CD DG44 medium. Place the flask(s) on an orbital shaker platform rotating at 130–135 rpm at 37°C, 80% relative humidity, and 8% CO2 2. On the day of transfection, harvest appropriate amount of cells (total of 1.
Transfecting CHO DG44 Cells with Neon® for two-subunit protein expression, continued Transfection procedure for expression of two-subunit protein, continued 9. Remove the Neon® Pipette from the Neon® Pipette Station and immediately transfer the samples from the Neon® Tip by pressing the push-button on the pipette to the first stop into the prepared T-75 flask(s) containing pre-warmed medium without antibiotics. Discard the Neon® Tip into an appropriate biological hazardous waste container. 10.
Selecting stable transfectants for two-subunit protein expression Introduction To obtain cell lines that produce high levels of your protein, first select for a pool of stably-transfected cells, in which the linearized pOptiVEC™ and pcDNA™3.3 constructs have integrated into the host cell genome. Perform the selection using complete CD OptiCHO™ Medium containing 500 µg/mL of Geneticin® Selective Antibiotic. Note that only cells that have been transfected with pOptiVEC™ and pcDNA™3.
Selecting stable transfectants for two-subunit protein expression, continued Selecting stable transfectants for two-subunit protein expression 48 hours after transfection, passage transfected CHO DG44 Cells (cGMP banked) in complete CD OptiCHO™ Medium containing 500 µg/mL of Geneticin® reagent to select for stably transfected clones. To passage cells: 1. Determine viable and total cell counts (see page 16). 2.
Assessing productivity Protein production Important Choosing a workflow To check for production of your protein during stable cell establishment, you may take an aliquot of growth media and perform SDS-PAGE, protein-specific ELISA, or the bioactivity assay of choice to determine that your cells are producing your protein of interest. When you have a pool of stably-transfected cells, freeze several aliquots of the pool using the procedure on page 18.
Assessing productivity, continued Points to consider When choosing a workflow, consider the amount of protein you wish to produce, your available resources, and the amount of time it will take to obtain your clonal, high-producing cell lines. Because MTX amplification produces a polyclonal population, you must always perform clone selection prior to scale-up. Additional cloning media, supplements, and other products may be purchased separately (page 70).
Genomic amplification by MTX addition Introduction Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folate transporter. In the cell, it is converted to a high molecular weight polyglutamate metabolite by folylpolyglutamate synthase, which binds to DHFR and inhibits its activity. If MTX is present in the medium, cells compensate by increasing the DHFR copy number in the genome to overcome inhibition by MTX.
Genomic amplification by MTX addition, continued One round of MTX amplification The productivity of each clone depends upon the integration locus of your expression construct, the response to amplification using MTX, and the nature of your protein. Depending on your protein production needs, your available time, and your resources, you may perform one round of MTX amplification at various concentrations (such as 50 nM, 100 nM, 250 nM, 500 nM, and 1 µM).
Clonal selection by limiting dilution Introduction Development of a CHO cell line for commercial production of a recombinant protein requires clonality of the final cell population. This is achieved by limiting dilution cloning (LDC). Before performing LDC, expand the stably transfected or MTX-amplified cells in CD OptiCHO™ medium supplemented with 8 mM glutamine and without any selection pressure for at least two passages. On the day of cloning, dilute the cells to seed 0.
Clonal selection by limiting dilution, continued Preparing cloning medium The procedure described below uses CD FortiCHO™ Medium as the cloning medium. One 50-mL conical tube is sufficient to seed approximately 200 wells at 1 cell/well. For greater numbers of wells or plates, adjust the volumes accordingly. 1. Thaw L-glutamine to be used in preparation of completed cloning medium. 2.
Clonal selection by limiting dilution, continued Cell counting and dilution 1. Label five 50-mL conical tubes “1” through “5”. 2. Pipette 5 to 10 mL of your transfected pool CHO DG44 cells into the 50-mL tube labeled “1”. 3. Determine the viable cells/mL accurately using your method of choice. 4.
Clonal selection by limiting dilution, continued Plating cells 1. After warming the cloning medium (step 4, page 38), remove it from the incubator and place it in the laminar flow hood. 2. Pipette 0.2 mL of the cell suspension from “Tube 5” (1,000 cells/mL) into the cloning medium. 3. Mix the cell suspension gently by inverting the tube 5 or 6 times and transfer it into a sterile reagent reservoir or trough. 4.
Clone scale-Up Introduction After isolating your clones of interest (previous section), transfer single-cell colonies from 96-well plates to 24-well plates, and then scale up the volume of cells every 3–7 days by transferring each clone into the next larger plate or vessel (i.e., 24-well plates to 6-well plates to T-25 flask and then to 125-mL shaker flasks). Note The total clone scale-up process from a 96-well plate to a 125-mL flask takes about 2–4 weeks, depending on the growth rate of each clone.
Methods for single-subunit protein expression One-page flowcharts Introduction The following pages contain flowcharts to aid you in your expression experiments. Each flowchart consists of an outline of the necessary steps and space for notes that you can then transfer to your laboratory notebook. Using the one-page flowcharts The page numbers by the experimental steps in the flowcharts are hyperlinked to detailed protocols.
Expressing single-subunit protein using pOptiVEC™ expression construct Introduction The flowchart below depicts the major steps to transfect suspension CHO DG44 Cells (cGMP banked) with the pOptiVEC™ construct containing your gene of interest (GOI) using the FreeStyle™ MAX Reagent.
Genomic amplification by MTX addition Introduction The flowchart below depicts the major steps to amplify the copy number of your gene of interest (GOI) using MTX (as methotrexate hydrate) for increased production of the protein of interest.
Clonal selection by limiting dilution (single-subunit protein) Introduction The flowchart below depicts the major steps to obtain a clonal cell line (i.e., derived from a single cell) for the production of a single-subunit protein by diluting the pool of stably transfected cells or the MTX-amplified cells to 0.5–2 cells per well in a 96-well plate containing cloning medium. In most cases, one of the cells forms a distinct colony that can later be scaled up.
Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression Introduction You will use FreeStyle™ MAX Reagent to transfect suspension CHO DG44 Cells (cGMP banked) with the pOptiVEC™ expression construct containing your gene of interest IMPORTANT! If you are expressing a single-subunit protein, you must generate your expression construct using the pOptiVEC™-TOPO® TA vector.
Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression, continued Recommendation Calculate the number of CHO DG44 Cells (cGMP banked) that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 95% viable before proceeding to transfection.
Transfecting CHO DG44 Cells with FreeStyle™ MAX Reagent for single-subunit protein expression, continued Transfection procedure using pOptiVEC™ expression construct Follow the procedure below to transfect CHO DG44 Cells (cGMP banked) in a 30-mL volume using your pOptiVEC™ expression construct. We recommend including negative controls (no DNA, no FreeStyle™ MAX Reagent) in your experiment to help you evaluate your results. 1.
Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression (optional) Recommendation Calculate the number of CHO DG44 Cells (cGMP banked) that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 95% viable before proceeding to transfection.
Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression, continued Preparing cells Transfection procedure for expression of single-subunit protein 1. 24 hours before transfection, passage CHO DG44 cells at 3 × 105 cells/mL in complete CD DG44 medium. Place the flask(s) on an orbital shaker platform rotating at 130–135 rpm at 37°C, 80% relative humidity, and 8% CO2 2. On the day of transfection, harvest appropriate amount of cells (total of 1.
Transfecting CHO DG44 Cells with Neon® for single-subunit protein expression, continued Transfection procedure for expression of single-subunit protein, continued 9. Remove the Neon® Pipette from the Neon® Pipette Station and immediately transfer the samples from the Neon® Tip by pressing the push-button on the pipette to the first stop into the prepared T-75 flask(s) containing pre-warmed medium without antibiotics. Discard the Neon® Tip into an appropriate biological hazardous waste container. 10.
Selecting stable transfectants for single-subunit protein expression Introduction To obtain cell lines that produce high levels of your protein, first select for a pool of stably-transfected cells, in which the linearized pOptiVEC™ construct has integrated into the host cell genome.
Assessing productivity Protein production To check for production of your protein during stable cell establishment, you may take an aliquot of growth media and perform SDS-PAGE, protein-specific ELISA, or the bioactivity assay of choice to determine that your cells are producing your protein of interest. IMPORTANT! When you have a pool of stably-transfected cells, freeze several aliquots of the pool using the procedure on page 18.
Assessing productivity, continued Points to consider When choosing a workflow, consider the amount of protein you wish to produce, your available resources, and the amount of time it will take to obtain your clonal, high-producing cell lines. Because MTX amplification produces a polyclonal population, you must always perform clone selection prior to scale-up. Additional cloning media, supplements, and other products may be purchased separately (page 70).
Genomic amplification by MTX addition Introduction Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folate transporter. In the cell, it is converted to a high molecular weight polyglutamate metabolite by folylpolyglutamate synthase, which binds to DHFR and inhibits its activity. If MTX is present in the medium, cells compensate by increasing the DHFR copy number in the genome to overcome inhibition by MTX.
Genomic amplification by MTX addition, continued One round of MTX amplification The productivity of each clone depends upon the integration locus of your expression construct, the response to amplification using MTX, and the nature of your protein. Depending on your protein production needs, your available time, and your resources, you may perform one round of MTX amplification at various concentrations (such as 50 nM, 100 nM, 250 nM, 500 nM, and 1 µM).
Clonal selection by limiting dilution Introduction Development of a CHO cell line for commercial production of a recombinant protein requires clonality of the final cell population, achieved by limiting dilution cloning (LDC). Before performing LDC, expand the stably transfected or MTXamplified cells in CD OptiCHO™ medium supplemented with 8 mM glutamine and without any selection pressure for at least two passages. On the day of cloning, dilute the cells to seed 0.5–2 cells per well in a 96-well plate.
Clonal selection by limiting dilution, continued Preparing cloning medium The procedure described below uses CD FortiCHO™ Medium as the cloning medium. One 50-mL conical tube is sufficient to seed approximately 200 wells at 1 cell/well. For greater numbers of wells or plates, adjust the volumes accordingly. 1. Thaw L-glutamine to be used in preparation of completed cloning medium. 2.
Clonal selection by limiting dilution, continued Cell counting and dilution 1. Label five 50-mL conical tubes “1” through “5”. 2. Pipette 5 to 10 mL of your transfected pool CHO DG44 cells into the 50-mL tube labeled “1”. 3. Determine the viable cells/mL accurately using your method of choice. 4.
Clonal selection by limiting dilution, continued Plating cells 1. After warming the cloning medium (step 4, page 58), remove it from the incubator and place it in the laminar flow hood. 2. Pipette 0.2 mL of the cell suspension from “Tube 5” (1000 cells/mL) into the cloning medium. 3. Mix the cell suspension gently by inverting the tube 5 or 6 times and transfer it into a sterile reagent reservoir or trough. 4.
Clone scale-up Introduction After isolating your clones of interest (previous section), transfer single-cell colonies from 96-well plates to 24-well plates, and then scale up the volume of cells every 3–7 days by transferring each clone into the next larger plate or vessel (i.e., 24-well plates to 6-well plates to T-25 flask and then to 125-mL shaker flasks). Note The total clone scale-up process from a 96-well plate to a 125-mL flask takes about 2–4 weeks, depending on the growth rate of each clone.
Appendix A: Troubleshooting Troubleshooting Culturing CHO DG44 Cells (cGMP banked) Observation No viable cells after thawing original vial The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiment. Reason Solution Cells not stored correctly Order new cell stock and store in liquid nitrogen. Keep in liquid nitrogen until thawing.
Troubleshooting, continued Culturing CHO DG44 Cells (cGMP banked) Observation Cells grow slowly The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiment. Reason Incorrect growth medium Solution • • Use pre-warmed CD DG44 Medium supplemented with 8 mM L-glutamine and 18 mL/L Pluronic® F-68. Do not use CD OptiCHO™ Medium to propagate DHFR-negative CHO DG44 Cells (cGMP banked).
Troubleshooting, continued Transfection Observation The table below lists some potential problems and possible solutions that may help you troubleshoot your transfection experiments. Reason Very few or no stably- Improperly cultured cells transfected cells obtained Solution • Exactly follow procedures as outlined in Thawing and Subculturing Cells (page 15). • • Thaw a new batch of early-passage cells. Do not add antibiotics during transfection.
Troubleshooting, continued Protein Expression The table below lists some potential problems and possible solutions that may help you troubleshoot your protein expression levels. Observation No or low protein detected in the supernatant after transient or stable transfection Reason Solution PCR primer does not contain Kozak translation initiation sequence Add a Kozak consensus site to the forward PCR primer, resynthesize your DNA and reclone.
Appendix B: Vectors Map and features of pOptiVEC™-TOPO vector Map The map below shows the elements of the pOptiVEC™-TOPO vector. The vector sequence is available from www.lifetechnologies.com or by contacting Technical Support (page 76).
Map and features of pOptiVEC™-TOPO® vector, continued Features The pOptiVEC™-TOPO® vector contains the following elements. Features have been functionally tested, and the vectors have been fully sequenced. Feature Benefit Full length human cytomegalovirus (CMV) immediate-early promoter/enhancer Allows efficient, high-level expression of your recombinant protein (Andersson et al., 1989; Boshart et al., 1985; Hennighausen & Fleckenstein, 1986; Nelson et al., 1987).
Map and features of pcDNA™3.3-TOPO® vector Map The map below shows the elements of the pcDNA™3.3-TOPO®vector. The vector sequence is available from www.lifetechnologies.com or by contacting Technical Support (page 76).
Map and features of pcDNA™3.3-TOPO® vector, continued Features The pcDNA™3.3-TOPO® vector contains the following elements. Features have been functionally tested, and the vectors have been fully sequenced. Feature Benefit Full length human cytomegalovirus (CMV) immediate-early promoter/enhancer Allows efficient, high-level expression of your recombinant protein (Andersson et al., 1989; Boshart et al., 1985; Hennighausen & Fleckenstein, 1986; Nelson et al., 1987).
Appendix C: Ordering information Accessory products Freedom™ DG44 Kit Products Many of the components supplied with the Freedom™ DG44 Kit are also available separately. Ordering information is provided below. For more information, go to www.lifetechnologies.com or contact Technical Support (see page 76). Item Amount Catalog no.
Accessory products, continued Additional products The products listed below may be used with the Freedom™ DG44 Kit. For more information, go to www.lifetechnologies.com or contact Technical Support (see page 76). Item Amount Catalog no. 7 1 × 10 cells R800-07 FreeStyle™ CHO Expression Medium 1000 mL 12651-014 Pluronic F-68, 10% 100 mL 24040-032 HT Supplement (100X) 50 mL 11067-030 ™ CD FortiCHO Medium 1000 mL A11483-01 Trypan Blue Solution, 0.
Appendix D: Safety Chemical safety WARNING! 72 GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: • Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials.
Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves.
Appendix E: Purchaser notification Limited Label License Information Limited Use Label License No. 335: Technology with CRO Rights This product and its use is the subject of one or more issued and/or pending U.S. and foreign patent applications owned or controlled by Life Technologies Corporation.
Limited Label License Information, continued Limited Use Label License No. 296: DG44 Cells Notice to Purchaser: The cells are sold under license from Lawrence and Gail Urlaub Chasin (phone (212) 854-4645 or email lac2@columbia.edu) for research purposes only and no license for commercial use is included herein.
Documentation and support Obtaining support Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to www.lifetechnologies.com/support and search for the Certificate of Analysis by product lot number, which is printed on the box. Obtaining SDS Safety Data Sheets (SDSs) are available at www.lifetechnologies.com/support.
References Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989) Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, 8222-8229 Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985) A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530 Cole, C. N., and Stacy, T. P.
Notes 78
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