FuGENE ®HD Transfection Reagent1.What this Product DoesNumber of Transfection ExperimentsIn 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 6,000wells in a 96-well plate or 1,000 wells in a 24-well plate.L Optimal expression depends upon experimental conditions includ-ing cell type, passage history, confluence, seeding protocol, com-plex 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.FormulationFuGENE ® HD Transfection Reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterile-filtered, and pack-aged 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 evenafter repeatedly opening the vial (at least five times over a two-month 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 Ϫ20°C, briefly warm it to 37°C to dissolve any precipitate. It should function normally; however, do not return it to the freezer.Special HandlingN Always bring to room temperature and mix FuGENE ® HD Transfec-tion Reagent prior to use (vortex for one second or use inversion).N Do not aliquot FuGENE ® HD Transfection Reagent from the originalglass 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 sur-faces.N Always dilute the reagent by pipetting directlyinto 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 RequiredAdditional 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 mixerFor 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 experi-ments)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 Transfec-tion Reagent vial•sterile polystyrene tubes or round-bottom 96-well platesCells 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 cellsApplicationFuGENE ® 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 .•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 .For the transient and stable transfection of animal and insect cellsCat. No. 04 709 691 0010.4 ml (up to 120 transfections)Version November 2007Cat. No. 04 709 705 001 1 ml (up to 300 transfections)Store at +2 to +8°CCat. No. 04 709 713 001 1)Mega-pack 5 × 1 ml (up to 1,500 transfections)Cat. No. 05 061 369 00110 ml (up to 3,000 transfections)Cat. No. 04 883 560 001Trial-pack1)The five vials are packaged together in one box with one pack insertFor life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.2.How to Use this Product2.1Before you BeginRequired Amount of FuGENE® HD Transfection ReagentFor 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 sec-tion 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 ConditionsMinimize 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 den-sity. 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% con-fluent 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 AdditivesIn 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 experi-ments. 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 FunctionOptimize 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.2.2Preparation of Cells for Transfection2.3Overview of Initial Transfection ExperimentAdherent and Suspension Cells in a Six-well Plate or 35-mm Culture DishFor 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 com-plex 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 com-monly 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/hdRatio OverviewPreparation 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.Cell Type ProcedureAdherentcellsOne day before the transfection experiment, trypsinizethe monolayer, adjust cell concentration, and plate thecells in the chosen cell-culture vessel. For most celltypes, plating 3 – 6 × 105 cells in 2 ml of medium in a35-mm culture dish (or six-well plate) overnight willachieve the desired density of Ͼ80% confluency atthe time of transfection. For cell lines with specialcharacteristics, such as contact-inhibited NIH/3T3cells, a lower plating density should be used. If usingculture plates of a different size, adjust the total num-ber of cells, starting volume of FuGENE® HD Transfec-tion Reagent, and the starting mass of DNA inproportion to the relative surface area (Table 2). SuspensioncellsUse freshly passaged cells at a concentration of 5×105/ml to 1 × 106/ml in 2 ml of medium in a 35-mmculture dish (or six-well plate). If using culture platesof a different size, adjust the total number of cells,starting volume of FuGENE® HD TransfectionReagent, and the starting mass of DNA in proportionto the relative volume (Table 2).Tubelabel(ratio)Diluent(␮l)FuGENE® HDTransfectionReagent (␮l)DNA(␮g)Comments3:210032Add the entire volume toa 35-mm culture dish oreach well of a six-wellplate, or 2 – 15 ␮l to eachwell of a 96-well plate.Suggested volumes fordifferent culture vesselsare included in Table 2. 4:2100425:2100526:2100627:2100728:2100822.4Transfection ProcedureNotes:L As with any experiment, include appropriate controls. Preparewells with cells that remain untransfected, cells with transfection reagent alone, and cells with DNA alone.L For stable transfection experiments, the complex-containingmedium should be left unchanged until the cells need to be pas-saged. At that time, include the appropriate selection antibiotics (G 418* or Hygromycin B*).L To prepare transfection complexes for different-sized containers orparallel 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-wellplates 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 optimaltotal amount of complex may vary with cell line, cell density, day of assay, and gene expressed.L After performing the optimization experiment where several ratioswere tested, select a ratio in the middle of the plateau for future experiments.2.5Cotransfection ExperimentsSuggestionsFor 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 plas-mid DNA should be equal to the amount of plasmid used in a single plasmid transfection.ᕡAllow FuGENE ® HD Transfection Reagent, DNA, anddiluent to adjust to +15 to +25°C. Vortex for one second or invert the FuGENE ® HD Transfection Reagent vial to mix.ᕢDilute DNA with appropriate diluent, for example, Opti-MEM IReduced Serum Medium, serum-free medium (without anti-biotics 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 celllines, try sterile water or serum-free medium as an alterna-tive diluent.ᕣPlace 100 ␮l diluent, containing 2 ␮g DNA into each of six ster-ile tubes labeled 3:2, 4:2, 5:2, 6:2, 7:2, and 8:2.Recommendation : Use sterile polystyrene tubes or round-bottom, 96-well plates to form the transfection complex.L Due to manufacturer variability with release agents for96well plates, we suggest using tissue culture treated 96well plates to reduce variablity.ᕤForm the transfection complex by adding FuGENE ® HDTransfection 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 with-out allowing contact with the walls of the plastic tubes.N To avoid adversely affecting transfection efficiency, do notallow 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.ᕥMix 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 rotat-ing 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.ᕦAdd the transfection complex to cell s: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 laboratorycell 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. ᕧIncubate 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 than10 – 30% cell death, refer to section 3, Troubleshooting and /fugene/hd2.6Parameters for Optimization2.7Transfection 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.2.8Guidelines for Preparing FuGENE® HD Transfection Reagent:DNA Complex for Various Culture Vessel SizesThe starting volume and mass to add to the different culture vessels is based upon preparing a 100-␮l transfection complex as described in sec-tions 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 cm2Suggested seeding density for suspension cells = 250,000 – 500,000 cells per mlTab. 2: Refer to the table below when setting up your transfection reac-tions. T hese 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 trans-fection. This must be determined empirically. For some cell lines, 60–80% con-fluency 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.TroubleshootingParameter to beoptimizedProcedureFuGENE® HD Transfection Reagent:DNA ratio 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 Transfec-tion Reagent: ␮g DNA).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 Transfec-tion Reagent.Amount of transfectioncomplex addedTry adding 200%, 150%, 75%, 50%, and 25% of the amount of 100-␮l transfection complex suggested in Table 2.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 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 min-utes). 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.Special tips for sensitive cell lines •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).Culture vessel Surfacearea(cm2)TotalvolumeofmediumSuggested seeding densitySuggested amount ofthe 100-␮l trans-fection complex toadd to each well (␮l)Final amount of FuGENE® HDTransfection Reagent (␮l) ineach well following addition ofsuggested amount of 100-␮ltransfection complexCells/wellAdherent cellsCells/wellSmall or suspensioncellsUsing the3:2 ratioUsing the8:2 ratio totalvolumevolume forlargerlow high low high96-well plate(1 well)0.30.110,00020,00025,00050,00050.150.424-well plate(1 well)1.90.550,000125,000250,000500,000250.752.012-well plate(1 well)3.8 1.0100,000250,000375,000750,00050 1.54.0 35-mm dish82200,000500,000500,0001,000,000100 3.08.06-well plate(1 well)9.42200,000600,000500,0001,000,000100 3.08.0 60-mm dish215500,0001,400,0001,250,0002,500,000250 1)7.520.0 10-cm dish55101,500,0003,500,0002,500,0005,000,000500 1)15.040.0 T-25 flask256700,0001,700,0001,500,0003,000,000300 1)9.024.0 T-75 flask75202,000,0005,000,0005,000,00010,000,000900 1)27.072.0Low trans-fection efficiency Poor quality orinsufficient quantityof nucleic acidsVerify the amount, purity, and sequence of nucleic acid.Perform a control transfection experiment with a commercially available transfection-grade plasmidpreparation.Chemical contaminants may be in the plasmid preparation. Avoid phosphate buffers until you havetested them in your system.L Endotoxins are reported to be cytotoxic to some very sensitive cell lines.Insufficient numberof cellsUse adherent cells that are at least 80% confluent. Low cell density results in fewer cells available totake up transfection complex, and excess complex may be cytotoxic; in addition, fewer cells yieldless protein.Too many cells orcells post log phaseWhen confluent cultures are subcultured, or cells are plated at too high a density, the cells fail to dividein the culture being transfected. This results in suboptimal expression.Suboptimal FuGENE®HD TransfectionReagent:DNA ratio,complex incubationtime, total amount oftransfection complexadded, or cell densityOptimize the FuGENE® HD Transfection Reagent:DNA ratio, complex incubation time, amount ofcomplex 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 transfectionreagent:␮g DNA) following the protocol in this pack insert (sections 2.3, 2.4) and doubling theamounts of all components.•As soon as the complexes are combined and mixed, add 10, 5, or 2.5 ␮l of each complex to one of3columns of cells in each 96-well plate (i.e., columns 2, 3, and 4). Leave all outer wells empty ascontrols.•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-complexincubation 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 theratio 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 andvisit /fugene/hdFuGENE® HD Trans-fection Reagent wasaliquotedCheck that FuGENE® HD Transfection Reagent is stored in the original container. If the reagent wasaliquoted into plastic containers, there is a high chance of inactivation. Make sure the reagent isimmediately mixed with the dilute DNA either by vortexing or pipetting up to 10 – 15 times.FuGENE® HD Trans-fection Reagent cameinto contact withplastic or wasinadequately mixedRepeat transfection, carefully pipetting FuGENE® HD Transfection Reagent directly into the serum-freemedium, being careful not to touch the sides of the container while adding the FuGENE® HD Transfec-tion Reagent to the diluted DNA. If the FuGENE® HD Transfection Reagent is added too gently, it maylayer on top of the medium, thus making contact with the plastic.Transfection complexwas formed in serum-containing mediumCheck original bottle of medium used for complex formation. Repeat experiment using new bottle ofOpti-MEM that does not contain any additives (e.g., serum, antibiotics, growth enhancers, heparin,dextran sulfate, etc.). Try forming the complex in sterile water or plain DMEM.Media and mediacomponentsDifferent media and media components may influence the level of transfection efficiency andsubsequent growth of the transfected cells, as well as expression of the recombinant protein. Some lotsof 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 seraand media. Check that the medium and/or serum is from the same lot that worked previously. Try newlots or a different vendor.Culture may becontaminated withmycoplasmaCultures contaminated with mycoplasma have been shown to have decreased transfection efficacy.Determine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Inconsistent results Ratio or amount oftransfection complexis at the edge ofperformance plateauInitial experiments should be completed to determine the ratios, amount of complex to be added, andlength of time for complex formation for optimal performance. In our experience, we have found the pla-teau to be relatively broad. We recommend that future experiments be performed with ratios, incubationtime, and amounts of complex that were in the middle of the plateau. If conditions are selected at theedge of the plateau, very small procedural differences may cause large differences in the resulting pro-tein expression. Increased consistency may be achieved by shifting parameters away from the edge ofthe plateau to the middle of the plateau.Transfection complexformation:timing,amounts, and ratioFormation of the complex involves a multifaceted interaction between the transfection reagent and DNAas well as biological parameters. Differences in any of the components or techniques may result ininconsistencies. If results do not meet your expectations, then repeat the optimization experimentselecting areas near the plateau found in previous experiments. For current experiments, determine ifyou should use a different ratio, length of time, or amount of complex for more consistent transfectionresults.Extensive testing of the FuGENE® HD Transfection Reagent is performed on two cell lines: one easy totransfect and one very difficult to transfect. All reagent lots must pass this rigorous testing before wemake it available to you. However, we cannot test all cell lines, media, sera, and vectors; in your labora-tory, you may find slight differences in the optimal ratio, amount of complex, or time for complex forma-tion for some lots of FuGENE® HD Transfection Reagent.Cells 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 transfectionexperiments, but for other cell lines, these changes have profound effects. Each cell type may have a dif-ferent optimal transfection condition. Optimal values for a single cell type may also change slightly withvector construct and type of protein expressed.Observation Possible cause RecommendationSigns of cytotoxicity Transfected protein iscytotoxic or isproduced at highlevelsReduced viability or slow growth rates may be the result of high levels of protein expression, as the cell’smetabolic resources are directed toward production of the heterologous protein. The expressed proteinmay also be toxic to the cell at the level expressed.To analyze cytotoxicity, prepare experimental controls as described below.Prepare extra control wells containing:ቢ Cells that are not transfectedባ Cells treated with DNA alone (e.g., without FuGENE® HD Transfection Reagent)ቤ Cells treated with FuGENE® HD Transfection Reagent alone (no DNA added)ብ Cells transfected with a non-toxic or secreted protein.Compare experimental transfected cells to cells in the control wells (described above). Considerrepeating the experiment with a secreted reporter gene such as SEAP, hGH, or a standard ␤-gal controlvector. Cells expressing SEAP should show little to no evidence of cytotoxicity.Too much transfec-tion complex fornumber of cellsIncrease the number of cells plated, and/or decrease the total amount of complex added to the cells. Trydifferent ratios and allow the complexes to form for different time intervals. Add different amounts ofcomplex; for example, make the complex as usual but add 75%, 50%, or 25% of the usual amounts toeach well. See Suboptimal FuGENE® HD:DNA Ratio in "Low transfection efficiency" section of this tablefor details or optimization protocol.Culture may becontaminated withmycoplasmaDetermine if culture is contaminated with mycoplasma; use the Mycoplasma Detection Kit* orMycoplasma PCR ELISA* to assess contamination.Cells may not behealthyAssess physiological state of cells and the incubation conditions (e.g., check incubator CO2, humidity,and temperature levels). Observe cells prior to each passage for morphology and absence of contami-nants. Make sure cells do not overgrow. Routinely passage cells prior to reaching confluency. Make surethat culture media and additives are within expiration date and have been stored properly.Diluent is toxic to thecellsDMEM is toxic to some insect cell lines. For these cells, prepare the transfection complex in sterilewater. You may also try forming the complex in the medium in which the cells are growing, providingthat the medium does not contain serum, heparin, or dextran sulfate.Plasmid preparationcontaminated withendotoxinEndotoxin is reported to be cytotoxic to sensitive cell lines.If above tests provenegative, FuGENE®HD TransfectionReagent may not beoptimal for your cells.Try FuGENE® 6 Transfection Reagent*, DOTAP Liposomal Transfection Reagent*, DOSPER LiposomalTransfection Reagent*, or X-tremeGENE Q2 Transfection Reagent*.High protein-expression levelsHigh expression levels of certain intracellular proteins (e.g., Green Fluorescent Protein [GFP]) may becytotoxic to some cell types. Cell proliferation, toxicity, and cell death may be monitored using Apoptosisand Cell Proliferation products from Roche Applied Science (visit /apoptosis for more information).Media and mediacomponentsTest 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 particularlyimportant if the transfected cells are allowed to continue to grow for 3 – 7 days to provide maximalprotein expression.。