Lipofectamine™ LTX Reagent - FAQs

I accidentally left my lipid reagent at room temperature. Can I still use it?

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

Can I use Lipofectamine LTX in RNAi experiments?

We recommend Lipofectamine 3000 for delivery of vector-based RNAi but for direct transfection of synthetic siRNA, we recommend using Lipofectamine RNAiMAX. For in vivo siRNA experiments, we recommend Invivofectamine 3.0 Reagent.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine LTX for transfection of suspension cells, other hard-to-transfect cells, and primary cells?

We recommend using Lipofectamine 3000 which demonstrates much higher transfection efficiency and protein expression over all other transfection reagents for suspension, hard-to-transfect, and primary cells. Review Lipofectamine 3000 data for over 60 cell lines here https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html. For highest transfection efficiencies, we recommend the Neon Transfection System for Jurkat (94%) and K562 (90%) cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine LTX for transfecting plasmid DNA into primary cells?

We have tested four primary cell lines: HuVEC (human ubilical vein endothelial cells), NHFF (normal human foreskin fibroblast), MJ90, and NDHF with transfection efficiencies between 5-25%. Instead, we recommend using Lipofectamine 3000 which shows highest transfection efficiency for all cell types and optimize by varying the DNA and lipid ratio. Alternatively, the Neon Transfection System shows 75-80% transfection efficiency. Please visit our Neon cell specific protocol database for more information: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html?icid=fr-neon-3

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use Lipofectamine LTX for delivery of plasmid DNA and siRNA?

Lipofectamine LTX was designed for delivery of plasmid DNA with minimal cytotoxicity. We do not recommend using it for siRNA delivery. We recommend using Lipofectamine 3000 with the P3000 Enhancer for co-transfection applications.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I choose between Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX?

Lipofectamine 3000 (Cat. No. L3000015is our best transfection reagent and we highly recommend this over all other reagents for plasmid DNA delivery for a broad range of cell types which includes:

- 60 cell lines (i.e., cancer, fibroblasts, neural, etc.)
- Easy-to-transfect cells
- Hard-to-transfect cells (i.e., neural, stem, blood-derived)
- Suspension cells
- Primary cells
- iPSCs
Please see our table of validated cell lines (https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html).

Lipofectamine 2000 (Cat. No. 11668019) and Lipofectamine LTX (Cat. No. 15338100) are alternative options that may work better for certain cell types. For addressing cytotoxicity issues, Lipofectamine 3000 is our most gentle reagent with significantly reduced cytotoxicity levels and improved transfection performance. Both Lipofectamine 3000 and Lipofectamine 2000 may be used for co-transfection (siRNA and plasmid DNA) experiments.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the difference between reverse transfection and forward transfection? What should I use?

In forward transfection, cells are seeded to appropriate confluence or cell density in wells or dishes, and the lipid-DNA complexes are added the next day. In reverse transfection, the transfection complexes are prepared inside the wells, after which cells and medium are added. Reverse transfection is faster to perform than forward transfection, and is the method of choice for high-throughput transfection. For non-high-throughput transfections, generally forward transfections have better efficiency for most cell types.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your transfection reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use antibiotics in the medium during transfection?

Antibiotics can be used in the medium for culturing of cell lines. However, we do not recommend using antibiotics in the transfection medium unless previously tested in the cell type and payload being transfected. This is because presence of antibiotics during transfection may adversely affect transfection efficiency (i.e., positively charged antibiotics binding to the DNA being transfected) and overall health of cells being transfected.

For stable transfection, we recommend waiting wait 24-48 hrs after transfection before adding selected antibiotics.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Is it necessary to use serum-free medium during lipid transfection?

It is not necessary to use serum-free medium during lipid transfection. However, it is critical to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum-containing medium. For optimal results with Lipofectin Transfection Reagent, we recommend performing transfection in medium without serum.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What kind of tubes should I use for making lipid:DNA complexes?

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products without issue.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

Which lipid transfection reagent would you recommend to use for my cell line?

We recommend using Lipofectamine 3000 Reagent (Cat. No. L3000015) for the delivery of plasmid DNA, Lipofectamine MessengerMAX Reagent (Cat. No. LMRNA001) for mRNA or short oligos, and Lipofectamine RNAiMAX Reagent (Cat. No. 13778150) for siRNA or miRNA. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What controls do you recommend that I use when performing a transfection?

To ensure optimal transfection success, we recommend including a positive transfection control and additional controls to confirm cell health and reagent quality.

For DNA transfection, we recommend using the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146). For siRNA transfection, we recommend using either the BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or Silencer Select GAPDH Positive Control siRNA (Cat. No. 4390850). For protein transfection, we recommend co-transfecting with an EmGFP mRNA such as the Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).

Cell health and reagent quality controls:
- Cells only
- Cells + DNA or RNA or protein only
- Cells + lipid reagent only
- Cells + Opti-MEM only
- Cells + positive control

Find additional tips, troubleshooting help, and resources within ourTransfection Support Center.

What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

Here are some points to consider:

1. Select the lipid reagent that is likely to result in highest transfection efficiency for your cell type, payload, and application. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent.
2. Optimize both lipid reagent and DNA quantities. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, but the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be between 50% to 80% confluency at the time of transfection (please refer to specific reagent manual for details). Cells should be in the mid-log growth phase. For better consistency and reproducibility of results between transfection experiments, accurately count your cells with either a hemocytometer or the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Confirm that the promoter and/or enhancer (any gene regulatory sequences) of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagent that has been frozen or stored at temperatures below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

For additional tips ,please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

What is the stability of your transfection reagents?

Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of shipment unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance.

Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine transfection reagents.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

I have tried several different transfection reagents and have failed to transfect my gene into my cell line of interest. Do you have any suggestions?

We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells. You may also consider using a viral-based system (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/viral-delivery-mammalian-expression.html) to deliver your gene into your mammalian cell line of interest.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

I see a small granular precipitate on my cells (microscopically) following addition of the transfection reagent:DNA complexes to my cells. Will it decrease the transfection efficiency?

It is normal to see some precipitate on the cells. However, a common reason for detecting unsually high precipitate on cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why do I see cytotoxicity after performing transfection with Lipofectamine 2000? Can you please help?

Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
3. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
4. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
5. Possible Cause: Complexes not thoroughly mixed in growth medium
   Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
6. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
7. Possible Cause: Antibacterial agents were used in growth medium during transfection
   Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
8. Possible Cause: Transfection reagent stored improperly
   Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
9. Possible Cause: Cationic lipid reagent was oxidized
   Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

I am getting very low transfection efficiency with Lipofectamine 2000. Can you please provide some troubleshooting tips?

Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:

1. Possible Cause: Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presence of serum
   Suggested Solution: Use serum-free medium for dilutions of plasmid DNA, siRNA, and transfection reagents. Note: we recommend using Opti-MEM | Reduced Serum Medium (Cat. No. 31985-062)to dilute Lipofectamine 2000 and DNA before complexing.
2. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µL) ratio of 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine2000 (µL) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
3. Possible Cause: Not enough plasmid DNA used for dilution or complex formation
   Suggested Solution: Verify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT DNA Assays Kits (Q33130, Q33120).
4. Possible Cause: Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPure Nucleic Acid Purification Kits.
5. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
6. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
7. Possible Cause: Inhibitors were present in medium
   Suggested Solution: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
8. Possible Cause: Problems with assay used to measure efficiency or expression
   Suggested Solution: Use a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
9. Possible Cause: Promoter-enhancer on vector is not recognized by the cell type
   Suggested Solution: Verify that the promoter-enhancer on your vector construct is compatible with the target cell type.
10. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
11. Possible Cause: Transfection reagent stored improperly
    Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.


Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

How do I perform a dose-response curve or kill curve?

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin, Hygromycin, and Zeocin take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of viral transduction over transfection?

Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main advantage of lipid-mediated transfection over calcium phosphate-mediated transfection?

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is the main difference between transient and stable transfection?

During transient transfection the exogenous DNA does not integrate into the host genome, as a result some DNA is lost with every subsequent cell division. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since up to hundreds of copies of the DNA may be delivered into the cell. In stable transfection, under antibiotic selection pressure, the DNA integrates into the host cell genome and is passed onto their daughter cells during cell division. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1-2 copies of the DNA may be integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is Lipofectamine 3000 the same as Lipofectamine 2000? What about Lipofectamine LTX, Lipofectamine PLUS, and Lipofectin?

All of our lipid reagents have different cationic-lipid formulations, each with unique properties and specific applications. Lipofectamine 3000 (Cat. No. L3000015) provides the best transfection performance with lowest cytotoxicity for plasmid DNA and RNAi delivery for all cell types. Lipofectamine LTX (Cat. No. 15338100) was formerly designed for delivery of plasmid DNA with minimal cytotoxicity. Lipofectamine PLUS is a discontinued transfection reagent, although the PLUS Reagent is available and sold separately (Cat. No. 11514-015). Lipofectin (Cat. No. 18292011) was originally launched in the late 1980s and is considered our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new users try Lipofectamine 3000 first for optimal performance and lowest toxicity.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do you estimate the amount of DNA using A260 readings? What does A260/A280 mean?

1 A260 unit (double stranded DNA in H2O) = 50 mg/mL. The extinction coefficient will change if DNA is diluted in a buffer other than H2O. This will change the value indicated above.

Sample calculation:

Volume of plasmid DNA sample = 100 mL

Dilution (1/20) = 25 mL of the sample in 475 mL H2O

A260 of diluted sample = 0.65

Note: For optimal results, make sure OD values are within 0.1 and 1.0.

Concentration of plasmid DNA sample = 0.65 x 50 mg/mL x 20 (dilution factor) = 650 mg/mL

Amount of plasmid DNA in sample = 650 mg/mL x 0.1 mL (sample volume) = 65 mg

An A260/A280 value that is between 1.8 and 2.0 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (i.e., phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are your recommendations with regards to endotoxin-free DNA for transfections?

For transfection-grade plasmid DNA, use either a PureLink HiPure or PureLink Expi Plasmid Purification Kit with plasmid purity equivalent to 2X CsCl gradients. For endotoxin-free DNA (< 9.1 EU/µg) use a PureLink Expi Endotoxin-Free Kit (Maxi, Mega, or Giga). Endotoxin-free DNA is recommended for sensitive applications such as transfection of primary cells and research on gene therapy for plasmid vaccines. For more information, please click here: http://www.thermofisher.com/us/en/home/brands/product-brand/purelink.html.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What types of molecules can be transfected using your lipid-based transfection reagents?

Our cationic lipid transfection reagents are used to transfect DNA (plasmids or oligonucleotides), siRNA (or miRNA), mRNA, or proteins. DNA delivered may be in the form of plasmids, cosmids, or even YAC clones as large as 600 Kb. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use your lipid reagents to co-transfect plasmids?

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 µg plasmid, use 0.5 µg of each of two co-transfected plasmids, or 0.25 µg of each of 4 co-transfected plasmids. When performing co-transfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Is there a place where I can find references from other researchers who have used your reagents?

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Where can I find cell line-specific transfection protocols?

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the transfection does not perform as expected, we recommend optimizing the conditions described in the product manual. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For more troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport).

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Why are my transfections not reproducible?

In general, transfection efficiency will show some degree of variability between transfection experiments and among replicates in the same transfection experiment. For better reproducibility, keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. We recommend preparing one master mix of the DNA/lipid complexes for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats. To further minimize the effects of transfection variability on data analysis, consider co-transfecting an internal normalization reference control such as beta-galactosidase or luciferase with the expression plasmid. Below are possible reasons for why your transfection results are not reproducible, along with suggested solutions:

1. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
2. Possible Cause: Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
   Suggested Solution: Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details. For well or plates sizes not listed in the scaling table, calculate the total surface area and estimate the -fold difference from the 24-well. Use this -fold difference to adjust for reagent volumes, payload quantities, and seeding densities.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Can I use the same amount of any transfection reagent for different cell lines?

No.The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.

The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Are cell density (% confluency) and passage number important considerations for transfection?

Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either descreasing or increasing the quantity of complexes added to the culture. We recommend using Lipofectamine 3000 since it shows the best flexibility for variable seeding density without showing cytotoxicity issues and maintains high protein expression. Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX/PLUS provide excellent transfection efficiencies at confluencies between 70 and 90%. Some toxicity may be observed at lower confluencies but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media. Lipofectamine RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What recommendations do you have for selecting a transfection reagent?

Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What are the different methods available for transfection?

There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

How do I choose between Lipofectamine 2000 and Lipofectamine LTX?

Lipofectamine LTX (Cat. No. 15338100) and Lipofectamine 2000 (Cat. No. 11668019) are both recommended for plasmid DNA transfections. However, if you notice increased cytotoxicity with Lipofectamine 2000, Lipofectamine LTX is the preferred reagent due to significantly reduced cytotoxicity levels and improved transfection performance in a number of hard-to-transfect cell lines.

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Can Lipofectamine LTX be used for transfecting primary cells?

We have tested four primary cell lines: HuVec, NHFF (Normal human foreskin fibroblast), MJ90 and NDHF. We have observed transfection efficiency in the range of 5-25%. We suggest optimizing transfection efficiency for your primary cell line by varying DNA and lipid ratio. If you are using any other primary cell line, please follow guidelines suggested in the product insert. Alternatively, Neon electroporation system and viral delivery methods are recommended.

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Can Lipofectamine LTX be used in transfection of Suspension cells and other hard to transfect cells?

We have tested transfection of suspension cells such as Jurkat and K562. We achieved transfection efficiency of less than 5% for each of them. We do not notice toxicity with Lipofectamine LTX reagent when used with these suspension cells. For other cell lines please follow the guidelines in the product insert. However, if you experience low transfection efficiency, then we recommend Neon electroporation system or Viral delivery.

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Can Lipofectamine LTX be used in RNAi experiments?

Lipofectamine LTX performs well with vector-based RNAi experiments since you are transfecting DNA. But for direct transfection of RNA molecules like siRNA and Stealth RNAi negative control transfections, we recommend Lipofectamine RNAiMAX.

Which lipid transfection reagent do you recommend for my cell line?

The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine LTX (Cat. No. 15338100) or Lipofectamine 2000 (Cat. No. 11668019) for plasmid transfection, and Lipofectamine RNAiMAX (Cat. No. 13778150) for siRNA transfection. For primary cells, Lipofectamine LTX with PLUS reagent (Cat. No. 15338100 and Cat. No. 11514-015) is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon electroporation system (Cat. No. MPK5000) usually works better compared to lipid transfection reagent.

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Are lipid transfection reagents fluorescent?

The intrinsic fluorescence of Lipofectamine 2000 and Lipofectamine under FITC conditions was examined and the results were as follows:
- Lipofectamine 2000 in serum-free conditions (with or without DNA) - weak whitish fluorescence.
- Lipofectamine 2000 in serum-containing conditions (with or without DNA) - medium white and red fluorescence.
- Lipofectamine & Lipofectamine PLUS (with or without DNA) - orange fluorescence.
- We haven't seen any significant green fluorescence attributable to the Lipofectamine.

These observations were made after transfection of CHO-K1 cells and by examining the live cells in PBS. Other lipid transfection reagents were not studied extensively on this issue, but it is very likely they will produce fluorescence in transfected cells.

Fluorescence from various media components may be where the problem lies. For example, riboflavin apparently fluoresces in the green wavelength and thus DMEM, which has a high riboflavin content, can be problematic in fluorescence experiments. Ham's F12 medium is 10x lower in riboflavin and is more suitable for fluorescence work.

One possible solution is to perform fluorescence imaging in PBS, rather than in culture medium. Also, check to make sure the cells are healthy and intact. Lysed cells or cells under stress may generate autofluorescent products.


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Is my lipid (Lipofectin, Lipofectamine, DMRIE-C, Lipofectamine 2000, Oligofectamine, Cellfectin II) supposed to be cloudy?

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin II and Lipofectamine 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

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Why do I see precipitates on the cells after transfection?

A small granular-like precipitate may be detected microscopically on the cells after transfection using cationic lipid. This is normal. The presence or absence of this precipitate is not indicative of the transfection efficiency. The precipitate can be caused by presence of excess EDTA or cationic lipid. Use DNA in water or, if in TE, use EDTA concentrations of <0.3 mM in the diluted DNA. Ensure concentrations of cationic lipid reagents do not exceed recommended amounts in complex formation.

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How can I improve transfection efficiency with cationic lipids?

1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
2. Optimize the cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluency at the time of transfection (for Lipofectamine 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

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Is the passage number of my cells important to consider when doing transfection?

In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.

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How do I scale up or down my transfection reaction?

The number of cells, DNA, lipid, and medium volumes should be scaled up proportionately to the surface area of the plate. For commonly used culture vessels, please refer to the information below regarding actual area and area relative to a 24-well plate well.

Vessel type, Area (cm2), Area Relative to 24-well
96-well, 0.3 cm2, 0.2
48-well, 0.7cm2, 0.4
24-well, 2 cm2, 1
12-well, 4 cm2, 2
6-well, 10 cm2, 5
35 mm, 10 cm2, 5
60 mm, 20 cm2, 10
100 mm, 60 cm2, 30
150 mm, 140 cm2, 70
T25, 25 cm2, 12.5
T75, 75 cm2, 37.5
T150, 150 cm2, 75
T162, 162 cm2, 81
T165, 165 cm2, 82.5
40-50 ml, 25 cm2, 12.5
250-300 ml, 75 cm2, 37.5
650-750 ml, 162-175 cm2, 81-87.5
900 ml, 225 cm2, 112.5


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How do cationic lipids compare with calcium phosphate in transfection efficiency?

For many cell types, higher efficiencies are observed with cationic lipids than with calcium phosphate. Also, cationic lipid data are more reproducible from experiment to experiment. Calcium phosphate is inexpensive however, but pH variation as little as 0.2 can reduce transfection efficiency significantly.

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Can lipid reagents be used to cotransfect plasmids?

Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 ug plasmid, use 0.5 ug of each of two cotransfecting plasmids, or 0.25 ug of each of 4, etc. When performing cotransfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

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What is the shelf life of the lipid transfection reagents?

Stored at 4°C in a sealed container, the lipids are stable for 12 months. Do not freeze the lipids. At 4°C with long term storage, due to evaporation concentration of lipid may vary, please briefly spin the contents before use. Add less lipid if you start noticing toxicity.

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What types of molecules can be transfected with cationic lipid reagents?

Lipid reagents can be used to transfect DNA, RNA, oligonucleotides and siRNA. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. Lipofectin and Lipofectamine 2000 Reagent have also been used to transfect cells with proteins (Beta galactosidase and T3 polymerase).

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Does the method of generating lipid-DNA complex affect transfection efficiency?

YES. Please follow the recommended procedure for each one of the reagents, found in the product manual.

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Can I use the same amount of any lipid reagent for transfection of my particular cell line?

No. The amount of lipid for each lipid reagent should be optimized for each cell line. Each lipid reagent has different composition/formulation, which will have different impact on each cell line. Therefore please optimize whenever you have a new lipid for each cell line.

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I have optimized my conditions for a particular cell line but I seem to be getting inconsistent results with my transfection. What factors contribute to this inconsistency?

Cells from a different passage number may behave differently. Also, if cells were sitting at confluence prior to plating for transfection, they may not transfect efficiently. To minimize such inconsistencies, passage the cells while they are still growing exponentially. Actively dividing cells transfect better.

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What kind of tubes can I use to form DNA:lipid complexes?

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products.

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Are there any guidelines for choosing a lipid transfection reagent?

It is best to optimize for your cells and application. Here are some basic guidelines:

- Lipofectamine LTX and PLUS Reagent: Minimal optimization, excellent efficiency with adherent eukaryotic cells DNA, difficult cell lines
- Lipofectamine Reagent: Adherent eukaryotic cells, DNA, oligonucleotides
- Lipofectin Reagent: Transfecting DNA in eukaryotic cell
- Cellfectin II: Insect cells
- DMRIE-C Reagent: DNA, RNA, suspension cells
- Oligofectamine: Oligonucleotides
- Lipofectamine RNAiMAX: siRNA, pre-miR, miRNA, anti-miR

NOTE: Please also visit our online Transfection Selection Tool to get specific recommendation for your cell line


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In the case of the 96-well protocol, Lipofectamine 2000 requires 320 ng DNA, whereas Lipofectamine PLUS, or Lipofectamine LTX requires up to 200 ng DNA. Does this mean that more DNA is needed to transfect efficiently when using Lipofectamine 2000?

Yes, you do need more DNA per well when using Lipofectamine 2000, because the optimal confluency of the cells is 90% (as compared to 50-80% confluent with Lipofectamine, or Lipofectamine LTX. Therefore you need more DNA in order to ensure that the maximum number of cells are transfected. With Lipofectamine 2000, we have found empirically that the higher confluence (~90%) of cells at the start of transfection improves transfection efficiency.

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