FuGENE HD Transfection Reagent

For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY. FuGENE HD Transfection Reagent For the transient and stable transfection of animal and insect cells Cat. No. 04 709 691 001 0.4 ml (up to 120 transfections) Version November 2007 Cat. No. 04 709 705 001 1 ml (up to 300 transfections) Store at +2 to +8 C Cat. No. 04 709 713 001 1) Mega-pack 5 1 ml (up to 1,500 transfections) Cat. No. 05 061 369 001 10 ml (up to 3,000 transfections) Cat. No. 04 883 560 001 Trial-pack 1) The five vials are packaged together in one box with one pack insert 1. What this Product Does Number of Transfection Experiments In a typical experiment using HeLa or COS-1 cells, 1 ml of FuGENE HD Transfection Reagent can be used to perform up to three hundred transfections in 35-mm tissue-culture dishes, using 3 l of reagent combined with 1 2 g DNA per well. This is equivalent to over 6,000 wells in a 96-well plate or 1,000 wells in a 24-well plate. L Optimal expression depends upon experimental conditions including cell type, passage history, confluence, seeding protocol, complex incubation time, serum batch, etc. The above amounts of reagents work well with HeLa or COS-1 cells. In other test systems, two- to three-fold higher amounts of reagent yield optimal levels of expression. Formulation FuGENE HD Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterile-filtered, and packaged in glass vials. It does not contain any ingredients of human or animal origin. Storage and Stability FuGENE HD Transfection Reagent is shipped at +15 to +25 C. Store FuGENE HD Transfection Reagent at +2 to +8 C, with the lid very tightly closed. The reagent is stable through the expiration date printed on the label when stored under these conditions. L FuGENE HD Transfection Reagent remains fully functional even after repeatedly opening the vial (at least five times over a twomonth period) as long as the vial is tightly recapped and stored at +2 to +8 C between uses. L Do not store FuGENE HD Transfection Reagent below 0 C. Components may precipitate and alter results. If you accidently place the reagent at 20 C, briefly warm it to 37 C to dissolve any precipitate. It should function normally; however, do not return it to the freezer. Special Handling N Always bring to room temperature and mix FuGENE HD Transfection Reagent prior to use (vortex for one second or use inversion). N Do not aliquot FuGENE HD Transfection Reagent from the original glass vials. Chemical residues in plastic vials can significantly decrease the biological activity of the reagent. Minimize the contact of undiluted FuGENE HD Transfection Reagent with plastic surfaces. 1107.04962095001➇ N Always dilute the reagent by pipetting directly into serum-free medium. Do not allow the FuGENE HD Transfection Reagent to contact the plastic walls of the tube containing the serum-free medium during the dilution step. N Do not use siliconized pipette tips or tubes. Additional Equipment and Reagents Required Additional reagents and equipment required to perform transfection assays using FuGENE HD Transfection Reagent, but not provided, include: General Laboratory Equipment standard cell culture equipment (e.g., biohazard hoods, incubators, microscope) standard pipetters and micropipetters vortex mixer For Plasmid Preparation purified plasmid stock (0.1 g/l 2.0 g/l) in sterile TE (10 mm Tris, 1 mm EDTA, ph 8.0) buffer or sterile water Genopure Plasmid Midi Kit*, Genopure Plasmid Maxi Kit*, or High Pure Plasmid Isolation Kit* can be used to prepare plasmid. For Verification of Vector Function assay appropriate for transfected gene G-418* or Hygromycin B* (optional; for stable transfection experiments) For Transfection-Complex Formation Opti-MEM I Reduced Serum Medium, water, or serum-free medium 24-well plate to serve as test tube rack for FuGENE HD Transfection Reagent vial sterile polystyrene tubes or round-bottom 96-well plates Cells Growing in Log Phase select subconfluent cultures in log phase for preparation of the cell cultures for transfection method to quantify cell number to reproducibly plate the same number of cells Application FuGENE HD Transfection Reagent is a multi-component reagent that forms a complex with DNA, then transports the complex into animal or insect cells. Benefits of FuGENE HD Transfection Reagent include: High transfection efficiency in many common cell types, including HeLa, NIH/3T3, COS-1, COS-7, CHO-K1, Hep G2, HEK-293, MCF7, and some insect cell lines. In addition, you will achieve excellent transfection efficiency in some cell lines (e.g., RAW) that are not transfected well by other reagents. Detailed transfection protocols and sample results are available at www.powerful-transfection.com. Demonstrates minimal cytotoxicity or changes in morphology when adequate numbers of cells are transfected, and eliminates the requirement to change media after the addition of transfection complex. Suitable for transient and stable transfection. Functions exceptionally well in the presence or absence of serum; eliminates the need to change media. To ensure the quality of cells to be transfected, Roche recommends using freshly-obtained, low-passage cell sines form ATCC. For more information please visit and bookmark www.atcc.org.

2. How to Use this Product 2.1 Before you Begin Required Amount of FuGENE HD Transfection Reagent For initial optimization experiments, transfect a monolayer of cells that is 80 90% confluent in a six-well culture dish, using 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 ratios of FuGENE HD Transfection Reagent (l) to DNA (g), respectively. For most cell types, these FuGENE HD Transfection Reagent:DNA ratios provide excellent transfection levels. L Subsequent optimization may further increase efficiency in your particular application. In addition to varying the volume of FuGENE HD Transfection Reagent, other parameters may be evaluated (see section 2.6, Parameters for Optimization, and section 3, Troubleshooting). Plasmid DNA It is critical to accurately determine the plasmid DNA concentration using 260-nm absorption (estimates of DNA content based on the intensity of gel bands are not sufficiently accurate). Determine the DNA purity using a 260 nm/280 nm ratio; the optimal ratio is 1.8. Prepare the plasmid DNA solution in sterile TE (Tris/EDTA) buffer or sterile water at a concentration between 0.1 g/l and 2.0 g/l. Cell Culture Conditions Minimize both intra- and inter-experimental variance in transfection efficiency by using cells that are regularly passaged, proliferating well (best when in a log-growth phase), and plated at a consistent density. FuGENE HD Transfection Reagent is different from FuGENE 6 Transfection Reagent regarding the optimal density of cells required for maximal expression with minimal negative effect; FuGENE 6 Transfection Reagent is formulated to work at low cell densities, whereas FuGENE HD Transfection Reagent is formulated to work at higher cell densities. Cells must be healthy and free of mycoplasma and other contaminants. L If you have used FuGENE 6 Transfection Reagent in the past, we suggest that you increase the plating density for initial tests using FuGENE HD Transfection Reagent. For most cell lines, use the reagent at cell-plating densities at least twice that used with FuGENE 6 Transfection Reagent to yield maximum protein expression. For most cell lines, cultures should be 80 90% confluent at the time of transfection. For contact-inhibited cell lines such as NIH/3T3, optimal results are obtained when cells are plated at lower densities. Other Media Additives In some cell types, antimicrobial agents (e.g., antibiotics and fungicides) that are commonly included in cell-culture media may adversely affect the transfection efficiency of FuGENE HD Transfection Reagent. If possible, exclude additives for initial experiments. Once high-efficiency conditions have been established, these components can be added back while monitoring your transfection results. Cell growth and/or transfection efficiency may be affected by variations in sera quality or media formulations. Verification of Vector Function Optimize transfection conditions with a known positive-control reporter gene construct prior to transfecting cells with a new vector construct: Determine transfection efficiency with a reporter gene assay (CAT*, -Gal*, Luciferase*, SEAP*, or hgh*). Sequence across the flanking vector insert regions to verify the integrity of your new construct. 2.2 Preparation of Cells for Transfection Cell Type Adherent cells Suspension cells Procedure One day before the transfection experiment, trypsinize the monolayer, adjust cell concentration, and plate the cells in the chosen cell-culture vessel. For most cell types, plating 3 6 10 5 cells in 2 ml of medium in a 35-mm culture dish (or six-well plate) overnight will achieve the desired density of 80% confluency at the time of transfection. For cell lines with special characteristics, such as contact-inhibited NIH/3T3 cells, a lower plating density should be used. If using culture plates of a different size, adjust the total number of cells, starting volume of FuGENE HD Transfection Reagent, and the starting mass of DNA in proportion to the relative surface area (Table 2). Use freshly passaged cells at a concentration of 5 10 5 /ml to 1 10 6 /ml in 2 ml of medium in a 35-mm culture dish (or six-well plate). If using culture plates of a different size, adjust the total number of cells, starting volume of FuGENE HD Transfection Reagent, and the starting mass of DNA in proportion to the relative volume (Table 2). 2.3 Overview of Initial Transfection Experiment Adherent and Suspension Cells in a Six-well Plate or 35-mm Culture Dish For initial optimization, test FuGENE HD Transfection Reagent:DNA ratios of 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2 (l for FuGENE HD Transfection Reagent, and g for DNA, respectively). The preparation of the complex for a single well of a six-well plate, or a 35-mm culture dish, is described in section 2.4. These ratios will function very well for commonly used adherent cells and suspension cells. For your particular cell line and culture conditions you may find that ratios of 9:2, 10:2, 11:2, or 12:2 result in even greater expression. Try these ratios if you find the highest expression levels in the 8:2 ratio well. N Prepare the transfection complex in diluent that does not contain serum (e.g., Opti-MEM I Reduced Serum Medium), even if the cells are transfected in the presence of serum. For some cell lines, the complex may be formed in DMEM or sterile water. L For additional optimization tips, see section 2.6 and visit /fugene/hd Ratio Overview Preparation of a transfection complex that is sufficient for a 35-mm culture dish, or one well of a six-well plate, at six different ratios: Tab. 1: Preparation of transfection complex for a 35-mm culture dish. Tube label (ratio) Diluent (l) FuGENE HD DNA Transfection (g) Reagent (l) Comments 3:2 100 3 2 Add the entire volume to 4:2 100 4 2 a 35-mm culture dish or each well of a six-well 5:2 100 5 2 plate, or 2 15 l to each 6:2 100 6 2 well of a 96-well plate. Suggested volumes for 7:2 100 7 2 different culture vessels 8:2 100 8 2 are included in Table 2. 2

2.4 Transfection Procedure Notes: Allow FuGENE HD Transfection Reagent, DNA, and diluent to adjust to +15 to +25 C. Vortex for one second or invert the FuGENE HD Transfection Reagent vial to mix. Dilute DNA with appropriate diluent, for example, Opti-MEM I Reduced Serum Medium, serum-free medium (without antibiotics or fungicides), or sterile water to a concentration of 2 g plasmid DNA/100 l Opti-MEM (0.02 g/l). L For insect cells, use sterile water as diluent. For other cell lines, try sterile water or serum-free medium as an alternative diluent. Place 100 l diluent, containing 2 g DNA into each of six sterile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2. Recommendation: Use sterile polystyrene tubes or roundbottom, 96-well plates to form the transfection complex. L Due to manufacturer variability with release agents for 96 well plates, we suggest using tissue culture treated 96 well plates to reduce variablity. Form the transfection complex by adding FuGENE HD Transfection Reagent to tubes containing diluted DNA: Pipet the FuGENE HD Transfection Reagent (3, 4, 5, 6, 7, or 8 l) directly into the medium containing the diluted DNA without allowing contact with the walls of the plastic tubes. N To avoid adversely affecting transfection efficiency, do not allow undiluted FuGENE HD Transfection Reagent to come into contact with plastic surfaces (such as the walls of the tube that contains the serum-free medium) other than pipette tips. Do not use siliconized pipette tips or tubes. Mix and incubate the transfection complex: Vigorously tap the tube or vortex for one to two seconds to mix the contents. If using a 96-well plate, place the plate on a rotating shaker for 5 10 seconds. Incubate the transfection reagent:dna complex for 15 minutes at room temperature. For some ratios and cell types, incubation is not necessary for optimal complex formation, while a longer incubation time is better for other cell types. Determine this for your particular cell line and the ratio you use. Add the transfection complex to cells: Remove culture vessel from the incubator. Removal of growth medium is not necessary. Add the transfection complex to the cells in a drop-wise manner or add below the surface of the medium. Swirl the wells or flasks to ensure distribution over the entire plate surface. Use of a rotating platform shaker for 30 seconds at low speed provides adequate mixing for 96-well plates. Once the FuGENE HD Transfection Reagent:DNA complex has been added to the cells, there is no need to remove and replace with fresh medium (as is necessary with some other transfection reagents). L In our experience, the exposure of most common laboratory cell types (COS-1, CHO-K1, HEK-293, HeLa, Hep G2, MCF-7) to the transfection complex until performance of the gene expression assay (24 48 hours later) does not affect the results. If you desire to transfect cells that are in serum-free medium during the transfection process, then replace the medium with serum-containing medium 3 8 hours after transfection, unless the cells normally grow in serum-free medium. Incubate cells and assay the results: Following transfection, incubate the cells for 18 72 hours prior to measuring protein expression. The length of incubation depends upon the transfected vector construct, the cell type being transfected, the cell medium, cell density, and the type of protein being expressed. After this incubation period, measure protein expression using an assay that is appropriate for your system. L If you observe low transfection levels or more than 10 30% cell death, refer to section 3, Troubleshooting and /fugene/hd L As with any experiment, include appropriate controls. Prepare wells with cells that remain untransfected, cells with transfection reagent alone, and cells with DNA alone. L For stable transfection experiments, the complex-containing medium should be left unchanged until the cells need to be passaged. At that time, include the appropriate selection antibiotics (G 418* or Hygromycin B*). L To prepare transfection complexes for different-sized containers or parallel experiments, proportionally change the quantity of all components according to the total surface area of the cell culture vessel being used (Table 2). L For ease-of-use when transfecting small volumes, as in 96-well plates containing 0.1 ml culture medium per well, prepare 100 l of transfection complex and add 2 15 l to each well depending upon the cell type. L The optimal ratio of transfection reagent:dna and the optimal total amount of complex may vary with cell line, cell density, day of assay, and gene expressed. L After performing the optimization experiment where several ratios were tested, select a ratio in the middle of the plateau for future experiments. 2.5 Cotransfection Experiments Suggestions For cotransfection experiments with FuGENE HD Transfection Reagent, maintain the same total reagent:total DNA ratio as that used for a single plasmid in your system. Thus, the total amount of the plasmid DNA should be equal to the amount of plasmid used in a single plasmid transfection. 3

2.6 Parameters for Optimization Parameter to be Procedure optimized FuGENE HD Transfection Form the transfection complex at several ratios: 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 10:2, and 12:2 (l FuGENE HD Transfection Reagent: g DNA). Reagent:DNA ratio In some systems, altering the ratio of FuGENE HD Transfection Reagent to DNA can increase the level of protein expression. L It has been reported that for some plasmid preparations, a ratio of 2:2 yielded optimal results. This is unusual and may reflect some property of the plasmid preparation rather than a characteristic of the FuGENE HD Transfection Reagent. Amount of transfection Try adding 200%, 150%, 75%, 50%, and 25% of the amount of 100-l transfection complex suggested in Table 2. complex added Number of cells plated Plating more cells will overcome negative growth effects of excess transfection complex. For cells with special growth characteristics, such as NIH/3T3 cells, do not use this as the first parameter for optimization. Incubation time for the transfection complex to form Special tips for sensitive cell lines Vary the length of incubation time for transfection-complex formation: add the complex to the cells immediately after the components are combined and mixed, and then at several intervals up to 40 minutes (i.e., 0, 15, 25, and 40 minutes). We have observed that in some cell lines, the transfection-complex incubation time tends to have no effect on results when using higher ratios; however, results using lower-ratio-complexes varied depending on the incubation time for complex formation. Reduce the time of exposure to the transfection complex (2 3 hours maximum), then replace the medium. Use the lower ratios, and allow the complex to form for a longer period of time (determine empirically for your cell line), then add lower amounts of the complex (50% or less of what was originally tested). 2.7 Transfection of Adherent Cells Adapted for Suspension Growth In some cases, adherent cells may be adapted for suspension growth, thus enabling the production of transiently transfected cells on a very large scale. HEK-293 cells grown in suspension in serum-free medium that did not contain heparin or dextran sulfate produced significant amounts of protein following transfection. 2.8 Guidelines for Preparing FuGENE HD Transfection Reagent:DNA Complex for Various Culture Vessel Sizes The starting volume and mass to add to the different culture vessels is based upon preparing a 100-l transfection complex as described in sections 2.3 and 2.4. For best results, prepare a 100-l complex at different ratios and add varying amounts of each ratio when optimizing. The amounts below are based on the 100-l complex as prepared in sections 2.3 and 2.4. Suggested seeding density for adherent cells = 30,000 70,000 cells per cm 2 Suggested seeding density for suspension cells = 250,000 500,000 cells per ml Culture vessel Surface area (cm 2 ) total volume Total volume of medium volume for larger Suggested seeding density Cells/well Adherent cells Cells/well Small or suspension cells low high low high Suggested amount of the 100-l transfection complex to add to each well (l) Final amount of FuGENE HD Transfection Reagent (l) in each well following addition of suggested amount of 100-l transfection complex Using the 3:2 ratio Using the 8:2 ratio 96-well plate (1 well) 0.3 0.1 10,000 20,000 25,000 50,000 5 0.15 0.4 24-well plate (1 well) 1.9 0.5 50,000 125,000 250,000 500,000 25 0.75 2.0 12-well plate (1 well) 3.8 1.0 100,000 250,000 375,000 750,000 50 1.5 4.0 35-mm dish 8 2 200,000 500,000 500,000 1,000,000 100 3.0 8.0 6-well plate (1 well) 9.4 2 200,000 600,000 500,000 1,000,000 100 3.0 8.0 60-mm dish 21 5 500,000 1,400,000 1,250,000 2,500,000 250 1) 7.5 20.0 10-cm dish 55 10 1,500,000 3,500,000 2,500,000 5,000,000 500 1) 15.0 40.0 T-25 flask 25 6 700,000 1,700,000 1,500,000 3,000,000 300 1) 9.0 24.0 T-75 flask 75 20 2,000,000 5,000,000 5,000,000 10,000,000 900 1) 27.0 72.0 Tab. 2: Refer to the table below when setting up your transfection reactions. These are suggested seeding densities and are media, passage level, laboratory, and cell-line dependent. It is critical that log phase cultures are selected for subculture for the transfection experiments, and that cultures are seeded at the proper density for the transfection experiment. Observe cultures and plate them so that the monolayer is 80 90% confluent at the time of transfection. This must be determined empirically. For some cell lines, 60 80% confluency is sufficient. However, a contact-inhibited cell line, such as NIH/3T3, should be plated at lower confluence due to its growth characteristics. 1) Scale up total volume for larger vessels. 3. Troubleshooting 4

Observation Possible Cause Low transfection efficiency Poor quality or insufficient quantity of nucleic acids Insufficient number of cells Too many cells or cells post log phase Suboptimal FuGENE HD Transfection Reagent:DNA ratio, complex incubation time, total amount of transfection complex added, or cell density FuGENE HD Transfection Reagent was aliquoted FuGENE HD Transfection Reagent came into contact with plastic or was inadequately mixed Recommendation Verify the amount, purity, and sequence of nucleic acid. Perform a control transfection experiment with a commercially available transfection-grade plasmid preparation. Chemical contaminants may be in the plasmid preparation. Avoid phosphate buffers until you have tested them in your system. L Endotoxins are reported to be cytotoxic to some very sensitive cell lines. Use adherent cells that are at least 80% confluent. Low cell density results in fewer cells available to take up transfection complex, and excess complex may be cytotoxic; in addition, fewer cells yield less protein. When confluent cultures are subcultured, or cells are plated at too high a density, the cells fail to divide in the culture being transfected. This results in suboptimal expression. Optimize the FuGENE HD Transfection Reagent:DNA ratio, complex incubation time, amount of complex added to cells, and cell density, according to the following procedure: Day before transfection: Prepare two 96-well plates of cells at high and low seeding densities (see Table 2 for suggestions). Day of transfection: Form 200 l of transfection complex at ratios of 2:2, 3:2, 4:2, 5:2, 6:2. 7:2, and 8:2 (l transfection reagent:g DNA) following the protocol in this pack insert (sections 2.3, 2.4) and doubling the amounts of all components. As soon as the complexes are combined and mixed, add 10, 5, or 2.5 l of each complex to one of 3 columns of cells in each 96-well plate (i.e., columns 2, 3, and 4). Leave all outer wells empty as controls. Continue to incubate the complexes at room temperature. After an additional 10 15 minutes, add 10, 5, or 2.5 l of each complex to the next 3 columns (5, 6, and 7) of cells in each 96-well plate. Continue to incubate the complexes at room temperature. After an additional 10 15 minutes, add 10, 5, or 2.5 l of each complex to the next 3 columns (8, 9, and 10) of cells in each 96-well plate. Assay the plates 1 2 days later. Select the ratio, amount of complex, and time of transfection-complex incubation that resulted in optimal expression. If optimal transfection occurs at the higher ratios, repeat this process using ratios of 6:2, 7:2, 8:2, 10:2, 12:2, and 14:2. Add 5, 10, and 15 l of complex. We have never successfully transfected cells using the ratio of 2:2, but it has been reported that some plasmid preparations transfect at this ratio. See section 2.6, Optimization of FuGENE HD Transfection Reagent:DNA ratio, for more information and visit /fugene/hd Check that FuGENE HD Transfection Reagent is stored in the original container. If the reagent was aliquoted into plastic containers, there is a high chance of inactivation. Make sure the reagent is immediately mixed with the dilute DNA either by vortexing or pipetting up to 10 15 times. Repeat transfection, carefully pipetting FuGENE HD Transfection Reagent directly into the serum-free medium, being careful not to touch the sides of the container while adding the FuGENE HD Transfection Reagent to the diluted DNA. If the FuGENE HD Transfection Reagent is added too gently, it may layer on top of the medium, thus making contact with the plastic. Transfection complex Check original bottle of medium used for complex formation. Repeat experiment using new bottle of was formed in serumcontaining Opti-MEM that does not contain any additives (e.g., serum, antibiotics, growth enhancers, heparin, medium dextran sulfate, etc.). Try forming the complex in sterile water or plain DMEM. Media and media components Culture may be contaminated with mycoplasma Different media and media components may influence the level of transfection efficiency and subsequent growth of the transfected cells, as well as expression of the recombinant protein. Some lots of sera have been reported to interfere with optimal transfection. Quality and/or lot-to-lot differences that affect transfection experiments have been noted in both sera and media. Check that the medium and/or serum is from the same lot that worked previously. Try new lots or a different vendor. Cultures contaminated with mycoplasma have been shown to have decreased transfection efficacy. Determine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* or Mycoplasma PCR ELISA* to assess contamination. 5

Observation Possible Cause Inconsistent results Ratio or amount of transfection complex is at the edge of performance plateau Transfection complex formation:timing, amounts, and ratio Cells Observation Possible cause Recommendation Signs of cytotoxicity Transfected protein is cytotoxic or is produced at high levels Too much transfection complex for number of cells Culture may be contaminated with mycoplasma Cells may not be healthy Diluent is toxic to the cells Plasmid preparation contaminated with endotoxin If above tests prove negative, FuGENE HD Transfection Reagent may not be optimal for your cells. High proteinexpression levels Media and media components Recommendation Initial experiments should be completed to determine the ratios, amount of complex to be added, and length of time for complex formation for optimal performance. In our experience, we have found the plateau to be relatively broad. We recommend that future experiments be performed with ratios, incubation time, and amounts of complex that were in the middle of the plateau. If conditions are selected at the edge of the plateau, very small procedural differences may cause large differences in the resulting protein expression. Increased consistency may be achieved by shifting parameters away from the edge of the plateau to the middle of the plateau. Formation of the complex involves a multifaceted interaction between the transfection reagent and DNA as well as biological parameters. Differences in any of the components or techniques may result in inconsistencies. If results do not meet your expectations, then repeat the optimization experiment selecting areas near the plateau found in previous experiments. For current experiments, determine if you should use a different ratio, length of time, or amount of complex for more consistent transfection results. Extensive testing of the FuGENE HD Transfection Reagent is performed on two cell lines: one easy to transfect and one very difficult to transfect. All reagent lots must pass this rigorous testing before we make it available to you. However, we cannot test all cell lines, media, sera, and vectors; in your laboratory, you may find slight differences in the optimal ratio, amount of complex, or time for complex formation for some lots of FuGENE HD Transfection Reagent. For consistent results, cells must be properly maintained. Cells change with passage level, passage conditions, media, and sera. For some cell lines, these changes have little to no effect on transfection experiments, but for other cell lines, these changes have profound effects. Each cell type may have a different optimal transfection condition. Optimal values for a single cell type may also change slightly with vector construct and type of protein expressed. Reduced viability or slow growth rates may be the result of high levels of protein expression, as the cell s metabolic resources are directed toward production of the heterologous protein. The expressed protein may also be toxic to the cell at the level expressed. To analyze cytotoxicity, prepare experimental controls as described below. Prepare extra control wells containing: Cells that are not transfected Cells treated with DNA alone (e.g., without FuGENE HD Transfection Reagent) Cells treated with FuGENE HD Transfection Reagent alone (no DNA added) Cells transfected with a non-toxic or secreted protein. Compare experimental transfected cells to cells in the control wells (described above). Consider repeating the experiment with a secreted reporter gene such as SEAP, hgh, or a standard -gal control vector. Cells expressing SEAP should show little to no evidence of cytotoxicity. Increase the number of cells plated, and/or decrease the total amount of complex added to the cells. Try different ratios and allow the complexes to form for different time intervals. Add different amounts of complex; for example, make the complex as usual but add 75%, 50%, or 25% of the usual amounts to each well. See Suboptimal FuGENE HD:DNA Ratio in "Low transfection efficiency" section of this table for details or optimization protocol. Determine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* or Mycoplasma PCR ELISA* to assess contamination. Assess physiological state of cells and the incubation conditions (e.g., check incubator CO 2, humidity, and temperature levels). Observe cells prior to each passage for morphology and absence of contaminants. Make sure cells do not overgrow. Routinely passage cells prior to reaching confluency. Make sure that culture media and additives are within expiration date and have been stored properly. DMEM is toxic to some insect cell lines. For these cells, prepare the transfection complex in sterile water. You may also try forming the complex in the medium in which the cells are growing, providing that the medium does not contain serum, heparin, or dextran sulfate. Endotoxin is reported to be cytotoxic to sensitive cell lines. Try FuGENE 6 Transfection Reagent*, DOTAP Liposomal Transfection Reagent*, DOSPER Liposomal Transfection Reagent*, or X-tremeGENE Q2 Transfection Reagent*. High expression levels of certain intracellular proteins (e.g., Green Fluorescent Protein [GFP]) may be cytotoxic to some cell types. Cell proliferation, toxicity, and cell death may be monitored using Apoptosis and Cell Proliferation products from Roche Applied Science (visit / apoptosis for more information). Test different media and optimize the level of each medium component for these cytotoxic effects. Although it is not usually necessary to remove the transfection complex following the transfection step, it may be necessary to feed your cells with fresh media for extended growth periods. This is particularly important if the transfected cells are allowed to continue to grow for 3 7 days to provide maximal protein expression. 6

4. Additional Information on this Product Quality Control Functional Analysis Cos 7 cells are transfected in 96 well plates with a reporter gene vector DNA, using 0.3 l FuGENE HD and 0.1 g DNA or 0.25 l FuGENE HD and 0.1 g DNA. Reporter gene activity is monitored via a colorimetric test against a reference lot. Activity must be 75% compared to a reference lot in at least one of the tested amounts, listed above. Cytotoxicity Analysis Cell growth is assessed using the Cell Proliferation Reagent WST-1* assay which is an indirect measure of cytotoxicity. The remainder of cells from functional analysis is tested for viability. Further Reading Linda B. Jacobsen et al. (2004) FuGENE 6 Transfection Reagent: The gentle power. Transfection of Mammalian Cells - Methods 33, 104-112. Roche Applied Science (2006a) Tips and tricks on how to improve transfection experiments. FuGENE HD Transfection Reagent, Application Note No. 1. Roche Applied Science (2006b) Identifying and solving transfection problems. FuGENE HD Transfection Reagent, Application Note No. 4. Roche Applied Science (2006c) Protocol for optimizing transfection of adherent cell lines. FuGENE HD Transfection Reagent, Application Note No. 3. Roche Applied Science (2007a) Transfection Control Experiment. FuGENE HD Transfection Reagent, Application Note No. 2. 5. Supplementary Information Changes to Previous Version Quality Control was modified New pack size added Concrete values have been added under Quality Control. Text Conventions To make information consistent and understandable, the following text conventions are used in this Instruction Manual: Text Convention Use Numbered stages labeled,, etc. Numbered instructions labeled,, etc. Asterisk * Stages in a process that usually occur in the order listed. Steps in a procedure that must be performed in the order listed. Denotes a product available from Roche Applied Science. Symbols Symbols are used in this Instruction Manual to highlight important information: Symbol L N Description Information Note: Additional information about the current topic or procedure. Important Note: Information critical to the success of the procedure or use of the product. Ordering Information Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our home page, and our Special Interest Site on transfection, www.powerful-transfection.com to get Application Protocols: Obtain information, including lists of successfully transfected primary or established cells and associated published references, cell-type specific protocols and proof data, instructions for special applications, technical tips on handling and optimization. Product Profiles: View detailed product description and readily access online purchasing. Transfected Cells Database: Browse regularly updated publications describing the use of Roche's Transfection Reagents. Search by cell type, transfected material, application, transfection reagent, or any combination of parameters to easily find a proven protocol for your specific application and cell type. Feedback: Add your evaluation results to the database of transfected cell types via an electronic questionnair. Product Pack Size Cat. No. Apoptosis and Cell Death Products Cell Proliferation Reagent WST-1 25 ml (2,500 tests) 11 644 807 001 Gene Knockdown Reagents X-tremeGENE sirna Dicer Kit 1 kit (10 reactions) 04 579 020 001 X-tremeGENE sirna Transfection Reagent 1 ml (400 transfections in a 24-well plate) 5 1 ml (2,000 transfections in a 24-well plate) 04 476 093 001 04 476 115 001 Mycoplasma Detection Reagents Mycoplasma Detection Kit 1 kit (25 tests) 11 296 744 001 Mycoplasma PCR ELISA 1 kit (96 reactions) 11 663 925 910 Plasmid Isolation Products Genopure Plasmid Midi Kit Genopure Plasmid Maxi Kit High Pure Plasmid Isolation Kit 1 kit (for up to 20 preparations) 1 kit (for up to 10 preparations) 1 kit (50 purifications) 1 kit (250 purifications) Protease Inhibitor Tablets and Lysis Reagents cpmplete 20 tablets in glass vials 3 x 20 tablets in glass vials 20 tablets in EASYpacks cpmplete, Mini 25 tablets in a glass vial 30 tablets in EASYpacks cpmplete, EDTA-free 20 tablets in a glass vial 3 x 20 tablets in glass vials 20 tablets in EASYpacks cpmplete, Mini, EDTA-free 25 tablets in a glass vial 30 tablets in EASYpacks cpmplete Lysis-B (2 ) (for bacterial cell lysis) cpmplete Lysis-B (2 ), EDTA-free (for bacterial cell lysis) cpmplete Lysis-M (for mammalian cell lysis) cpmplete Lysis-M, EDTA-free (for mammalian cell lysis) 1 kit (100 ml lysis reagent and 20 cpmplete Protease Inhibitor Cocktail Tablets) 1 kit (100 ml lysis reagent and 20 cpmplete, EDTAfree Protease Inhibitor Cocktail Tablets) 1 kit (200 ml lysis reagent and 20 cpmplete Protease Inhibitor Cocktail Tablets) 1 kit (200 ml lysis reagent and 20 cpmplete, EDTAfree Protease Inhibitor Cocktail Tablets) 03 143 414 001 03 143 422 001 11 754 777 001 11 754 785 001 11 697 498 001 11 836 145 001 04 693 116 001 11 836 153 001 04 693 124 001 11 873 580 001 05 056 489 001 04 693 132 001 11 836 170 001 04 693 159 001 04 719 930 001 04 719 948 001 04 719 956 001 04 719 964 001 Reporter Gene Assays CAT ELISA 1 kit (192 tests) 11 363 727 001 -Gal Reporter Gene Assay, chemiluminescent 1 kit (500 assays, microplate format, 250 assays, tube format) 11 758 241 001 -Gal ELISA 1 kit (192 tests) 11 539 426 001 hgh ELISA 1 kit (192 tests) 11 585 878 001 Luciferase Reporter Gene Assay, high sensitivity SEAP Reporter Gene Assay, chemiluminescent Selection Antibiotics G-418 20 ml 100 ml 200 assays 1,000 assays 1 kit (500 assays, microplate format, or 250 assays, tube format) 11 669 893 001 11 814 036 001 11 779 842 001 04 727 878 001 04 727 894 001 7

Product Pack Size Cat. No. Hygromycin B 1 g (20 ml) sterile filtered 10 843 555 001 Transfection Reagents FuGENE 6 Transfection Reagent Western Blotting Reagents Lumi-Light PLUS Western Blotting Kit (Mouse/Rabbit) Lumi-Light PLUS Western Blotting Substrate 0.4 ml (120 transfections) 1 ml (300 transfections) Multi-pack 5 1 ml (1,500 transfections) 1 Mega-pack 5 1 ml (1,500 transfections) 10 ml (3,000 transfections) 11 815 091 001 11 814 443 001 11 815 075 001 11 988 387 001 05 061 377 001 1 kit (1,000 cm 2 membrane) 12 015 218 001 100 ml (1,000 cm 2 membrane) 12 015 196 001 PVDF Western Blotting Membranes 1 roll (30 cm 3.00 m) 03 010 040 001 Western Blocking Reagent, Solution 100 ml (10 blots, 100 cm 2 ) 6 100 ml (60 blots, 100 cm 2 ) 11 921 673 001 11 921 681 001 Notice to Purchaser Purchaser represents and warrants that it will use FuGENE HD Transfection Reagent purely for research purposes. Transfected cells, materials produced, and any data derived from the use of FuGENE HD Transfection Reagent, may be used only for the internal research of Purchaser whether Purchaser is a for-profit or a not-for-profit organization. Under no circumstances may FuGENE HD Transfection Reagent be used by Purchaser or any third party for a commercial purpose unless Purchaser has negotiated a license for commercial use with Fugent, LLC (contact information: License@FugentLLC.com). For purposes of the foregoing sentence, commercial purpose shall mean use of FuGENE HD Transfection Reagent for profit or commercial gain. By using FuGENE HD Transfection Reagent, Purchaser agrees to be bound by the above terms. If Purchaser wishes not to be bound by these terms, Purchaser agrees to return the FuGENE HD Transfection Reagent to Roche Diagnostics for a full refund. Trademarks CpMPLETE, GENOPURE, HIGH PURE, and X-TREMEGENE are trademarks of Roche. FuGENE is a registered trademark of Fugent, L.L.C., USA. Geneticin and Opti-MEM are registered trademarks of Invitrogen Corporation. The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection. Other brands or product names are trademarks of their respective holders. Contact and Support To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at: /support To call, write, fax, or email us, visit the Roche Applied Science home page,, and select your home country. Countryspecific contact information will be displayed. Use the Product Search function to find Pack Inserts and Material Safety Data Sheets. Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany