Lipid-Based Transfection Support—Getting Started

Our cationic lipid transfection reagents can be used to transfect DNA, siRNA, Stealth RNAi™ siRNAs, mRNA, Dicer-generated siRNA pools, or plasmids containing shRNA cassettes. Oligonucleotides, proteins, and RNA can also be transfected. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. 

Please refer to the Transfection reagent selection guide to make the right choice. 

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 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 that 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.

In general, our recommended controls include:

• Cells only
• Cells + DNA or RNAi only
• Cells + lipid reagent only
• Cells + GFP plasmid positive control

These controls are important as they check cell health, reagent toxicity, and reagent function simultaneously.

Cell line–specific transfection protocols can be found here. 

If you do not find a cell line–specific protocol or if the protocol does not perform as expected, we recommend testing the conditions described in the protocol supplied with the product to determine the optimal protocol. 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 require additional optimization of the protocol to achieve the same result. 

We recommend using Lipofectamine™ 3000 Reagent or delivery of plasmid DNA, Lipofectamine™ MessengerMAX™ Reagent for delivery of mRNA or short oligos, and Lipofectamine™ RNAiMAX Reagent for delivery of siRNA/miRNA.

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 contact techsupport@thermofisher.com for further assistance with optimizing your protocol.

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

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

Yes. Cell density will affect transfection performance. Lipofectamine® 3000, Lipofectamine® 2000, and Lipofectamine® LTX/PLUS provide excellent transfection performance at confluencies between 70 and 90%, while some toxicity may be observed at confluencies lower than this. 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 for proper guidelines for culturing and passaging cells.

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.

Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection.We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

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. 

We recommend using Lipofectamine™ RNAiMAX Reagent for delivery of siRNA into all cell types. It has been specifically developed for siRNA transfection while providing high transfection efficiency with minimal cytotoxicity. As a result, less optimization is necessary. For vector DNA–based RNAi applications, we recommend Lipofectamine™ 3000 Reagent with the P3000™ Enhancer Reagent.

To prepare endotoxin-free DNA, we provide a number of nucleic acid purification kits, the PureLink® HiPure Purification Kits, in Mini, Midi, Maxi, Mega, and Giga sizes. These kits contain a patented anion-exchange resin to purify plasmid DNA to a level equivalent to 2X CsCl gradients. For more information, please click here. 

Expression in transiently transfected clones is typically higher because transiently transfected cells have a higher copy number of the gene (hundreds per cell). 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.

Please see the chart below:

Note: To determine the optimal concentration to use for stable selection, we recommend performing 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.

In forward transfection, cells are plated in wells, and the transfection complex is generally prepared and 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.

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 mg plasmid, use 0.5 mg of each of two co-transfected plasmids, or 0.25 mg 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.

• Lipofectamine™ 3000 & P3000™ Reagents are of a proprietary formulation.

• Lipofectamine™ 2000 Reagent is a proprietary formulation.

• Lipofectamine™ MessengerMAX Reagent is a proprietary formulation.

• Lipofectamine™ RNAiMAX Reagent is a proprietary formulation.

• Lipofectamine™ LTX Reagent is a proprietary formulation.

• Lipofectin™ Reagent is a 1:1 (w/w) formulation of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleolyphosphatidylethanolamine (DOPE).

• Lipofectamine™ Reagent is a 3:1 (w/w) formulation of the polycationic lipid 2.3-dioleyloxy-N(2(sperminecarboxamido)ethyl)-N, N-dimethyl- 1-propanaminium trifluoroacetate (DOSPA), and DOPE.

• PLUS™ Reagent is a proprietary formulation.

• DMRIE-C Reagent is a 1:1 (M/M) liposome formulation of the cationic lipid DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol.

• Cellfectin™ II Reagent is a proprietary formulation. 

• Lipofectamine™ 3000 Reagent results in the highest efficiency of plasmid DNA delivery into most cell types. It also works well for siRNA transfection and co-transfection of plasmid DNA with siRNA.
• Lipofectamine™ 2000 Reagent works well with most adherent cells as well as difficult-to-transfect cells. It can be used for transfection of plasmid DNA, siRNA, and co-transfection of plasmid DNA with siRNA.
• Lipofectamine™ MessengerMAX Reagent is recommended for transfection of mRNA or short oligos.
• Lipofectamine™ RNAiMAX provides the best results when transfecting siRNA/miRNA.
• Cellfectin™ II Reagent is recommended for transfection of insect cells.

Lipofectamine® 3000 is our best transfection reagent and is highly recommended for plasmid DNA delivery for a broad range of cell types that includes:

• >60 cell lines (i.e., cancer, fibroblasts, neural, etc.)
• Easy-to-transfect cells
• Hard-to-transfect cells (i.e., neural, stem, blood)
• Suspension cells
• Primary cells
• iPSCs

Please see our table of validated cell lines.

Lipofectamine® 2000 and Lipofectamine® LTX 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.

These are all different cationic-lipid formulations. Lipofectamine® 3000 provides the best transfection performance for both plasmid DNA and siRNA delivery over the broadest range of cell types.  Lipofectamine® 2000 is our earlier reagent that also works for delivery of both plasmid DNA and siRNA. Lipofectamine® LTX was 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® was originally launched in the late 1980s and is considered to be our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new customers try Lipofectamine® 3000 first for optimal performance and lowest toxicity. 

For getting successful co-transfection of plasmid DNA and siRNA, we recommend using high-quality DNA, a validated siRNA against the gene of interest, and log-phase growing cells. The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection:

1. To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, both the plasmid DNA and siRNA can be co-transfected using Lipofectamine® 3000 with the P3000™ enhancer. For the amounts of plasmid DNA, siRNA, Lipofectamine® 3000, and P3000™ enhancer to use for co-transfection, please refer to the manual and use the same amounts as recommended for individual transfection of plasmid DNA and siRNA.
2. To study the effect of siRNA knockdown of an endogenous gene, we recommend transfecting the siRNA first using Lipofectamine® RNAiMAX. 4–48 hours after delivery of siRNA, the plasmid DNA can be transfected using Lipofectamine® 3000. The time of post-transfection delivery of plasmid DNA may need to be optimized based on the half-life of the protein to be knocked down.
Once the appropriate method of delivery is determined based on the application, transfection should be optimized for DNA, siRNA, and transfection reagent doses. For transfecting HEK293 cells in 96 wells, we typically use these amounts per well:

• DNA: 0.1–0.2 μg
• siRNA: 1–3 pmoles
• Lipofectamine® 3000: 0.1–0.3 μL

We offer the Lipofectamine® MessengerMAX™ Transfection Reagent for transfection of mRNA, which offers up to 5X the efficiency of DNA transfection reagents in neurons and primary cells.

We recommend starting with Lipofectamine® 3000 Transfection Reagent using plasmid DNA; if that doesn’t work, we recommend trying Lipofectamine® MessengerMAX™ Transfection Reagent with mRNA.

Lipofectamine® 3000 Transfection Reagent was specifically designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cells. It works well for plasmid DNA transfection, siRNA transfection, and co-transfection of plasmid DNA with siRNA.

Yes, Lipofectamine® 3000 Transfection Reagent is an animal origin–free (AOF) product.

You can use Lipofectamine® 3000 for transfection of suspension cells, hard-to-transfect cells, and primary cells. Please see data here. However, if you experience low transfection efficiency, we recommend the Neon® Transfection System or the ViraPower™ Lentiviral Delivery Systems.

Yes, Lipofectamine® 3000 can be used to transfect primary cells including neurons, stem cells, and blood cells as shown here.

No, there are no cell-specific protocols as this reagent works well for a wide range of cell types. The optimization protocol in the manual provides for both a low and high dose option for Lipofectamine® 3000, where both dose types show comparable transfection efficiencies.

Lipofectamine™ 3000 works well for delivery of vector-based RNAi and synthetic siRNA, as well as co-transfection of siRNA with plasmid DNA. However, Lipofectamine™ RNAiMAX is our best transfection reagent for synthetic siRNA. For in vivo siRNA experiments, we recommend Invivofectamine™ 3.0 Reagent. 

For standard non-modified siRNA, Silencer® siRNA, Stealth RNAi™ siRNA, Silencer® Select siRNA, Ambion® Pre-miR Precursors, mirVana™ miRNA Mimics, Ambion® Anti-miR Inhibitors, and mirVana™ miRNA Inhibitors, use only the Lipofectamine® 3000 Reagent for efficient delivery to the cytoplasm. There is no need to use the P3000™ Reagent.

For plasmid DNA, vector-based BLOCK-iT™ shRNA or miRNA, ViraPower™ HiPerform™ lentiviral expression vectors, or GeneArt® CRISPR nuclease vectors, Lipofectamine® 3000 reagent used in combination with the P3000™ Reagent ensures for efficient delivery to the nucleus.

To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, both the plasmid DNA and siRNA can be co-transfected using Lipofectamine® 3000 with the P3000™ enhancer. Please use the same amounts of plasmid DNA, siRNA, Lipofectamine® 3000, andP3000™ enhancer as suggested in the manual for individual transfection of plasmid DNA and siRNA.

Lipofectamine® 2000 CD is a 100% synthetic (chemically defined) version of Lipofectamine® 2000. Thus, it is animal origin–free.

Lipofectamine® 2000:DNA complexes are stable for 6 hours at room temperature when the complex is prepared in Opti-MEM® medium. Use of other media for complex formation may decrease stability.

You can co-transfect plasmids and siRNA using Lipofectamine® 2000. Please click here for protocol information. 

Lipofectamine® LTX was designed for delivery of plasmid DNA with minimal cytotoxicity. We do not recommend using it for siRNA delivery. 

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 the DNA and lipid ratio. Our recommendation is to use Lipofectamine® 3000, which works well for a broad range of cell types. Alternatively, the Neon® Transfection System and lentiviral delivery methods are recommended.

We have tested transfection of suspension cells such as Jurkat and K562 usingLipofectamine® LTX. We achieved transfection efficiency of less than 5% for each of them. We recommend using Lipofectamine® 3000 for transfection of suspension cells, hard-to-transfect cells, and primary cells. Please see data here. However, if you experience low transfection efficiency, we recommend the Neon® Transfection System or the ViraPower™ Lentiviral Delivery Systems.

Lipofectamine™ LTX works well 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. 

PLUS™ Reagent is a proprietary reagent for pre-complexing DNA. It enhances cationic lipid-mediated transfection of DNA into many cultured eukaryotic cells when used in conjunction with a transfection reagent. Use PLUS™ Reagent to enhance transfection results with Lipofectamine® Reagent, Lipofectamine® LTX Reagent, Lipofectin® Reagent, Cellfectin® Reagent, and Oligofectamine™ Reagent. Lipofectamine® LTX is offered in combination with the PLUS™ Reagent in Cat. Nos. A12621, 15338030, and 15338100. PLUS™ Reagent is also offered as a stand-alone product (Cat. No. 11514015). We do not recommend using the PLUS™ Reagent with Lipofectamine® 2000 or Lipofectamine® 3000. 

No, it is important to dilute the DNA into medium and mix before adding the PLUS™ Reagent or the DNA may precipitate.

The Lipofectamine™ MessengerMAX™ Transfection Reagent has been specifically designed for transfection of mRNA into cells. It offers up to 5X the transfection efficiency of DNA transfection reagents in neurons and a broad spectrum of primary cells. This is possible because our novel lipid nanoparticle technology is optimized to deliver the highest amount of mRNA possible without the nuclear entry step that is required with DNA. The added advantage is that protein expression is faster with no risk of genomic integration.

Lipofectamine™ MessengerMAX™ Reagent is an excellent reagent for co-delivery of Invitrogen™ GeneArt™ CRISPR Cas9 mRNA with in vitro transcribed gRNA for genome editing purposes. Up to 10X higher cleavage efficiency can be obtained using mRNA CRISPRs. Lipofectamine™ MessengerMAX™ Reagent has the added benefit of flexibly delivering short dsDNA or HDR templates (0.5-1 kb) which can be ordered through the Invitrogen™ GeneArt™ Strings services.

No, the mMESSAGE mMACHINE® T7 ULTRA Transcription Kit (Cat. No. AMB1345) needs to be purchased separately.

We have validated the TriLink EGFP mRNA control (Cat. No. L-6101) [SA1] in-house.

Lipofectamine® MessengerMAX™ Transfection Reagent has a very simple one-tube protocol. The mRNA can either be transcribed using the mMESSAGE mMACHINE® ULTRA T7 Transcription Kit or can be purchased pre-made (e.g., GeneArt® CRISPR Nuclease mRNA, Cat. No. A25640).

Yes, Lipofectamine® MessengerMAX™ Transfection Reagent can deliver plasmid DNA in combination with mRNA (i.e., in CRISPR); however, Lipofectamine® 3000 Transfection Reagent is optimized for plasmid DNA delivery and delivers superior DNA transfection performance.

There are many advantages to using mRNA vs. DNA for transfection, and they are cell line–specific:

• A much higher level of transfection efficiency: If you are working with a difficult-to-transfect cell type, where DNA transfection yields less than 30% efficiency, transfecting an mRNA alternative can provide up to 80% transfection efficiency. Part of the hurdles that are encountered with DNA delivery has to do with the multiple steps required during transfection. In order to have a protein expressed in a cell, typically DNA is transfected, enters the nucleus, is transcribed to mRNA, is exported from the nucleus, and is translated into a protein in the cytoplasm. When delivering mRNA directly, the mRNA is present in the cytoplasm and ready for immediate translation to protein. However, if you are currently satisfied with the level of transfection achieved in the cell line that you are working with, there is no need to switch to mRNA.

• It is a footprint-free method with no risk of genomic integration: mRNA transfection is transient and does not enter the nucleus or pose a risk of integrating with the cellular host DNA, and is currently being researched for possible vaccine replacement and disease model development.

Additionally, transfection of mRNA with the newly developed Lipofectamine® MessengerMAX™ Reagent provides higher efficiency in a wider range of cell types (e.g., primary neurons, primary hepatocytes, primary keratinocytes, primary fibroblasts, iPS cells, hNSC, mESC, Raw 264.7, SH-SY-5Y, HT-29). This is a result of its ability to deliver the highest amount of mRNA independent of the cell model being used.

No, there are no specific medium requirements or restrictions when using mRNA for transfection. The only recommendation is to ensure that there is no serum or antibiotics present during the complexation process of the lipid and mRNA within the transfection protocol. We use Opti-MEM® I Reduced Serum Medium in our transfection protocol. The delivery of mRNA does not require a medium change after transfection. Our common practice is to not change medium within the first 24 hours following transfection, so as to minimize handling of cells.

Yes, transfection with mRNA results in faster and more immediate translation of protein and therefore, faster expression. We visually see expression of GFP in some cell lines as fast as 90 minutes after transfection. Additionally, transfection of mRNA with Lipofectamine® MessengerMAX™ Reagent also provides prolonged duration of expression (GFP expression lasting for 5 days post-transfection), due to its ability to protect mRNA from degradation during transfection.

The best reagent to transfect mRNA into cells is Lipofectamine™ MessengerMAX™ Transfection Reagent because it has been shown to further protect the mRNA molecules from degradation and has been shown to deliver the highest amount of mRNA into a wide variety of cell types. Please visit this page for more information. For tissues or small animal models, the best reagent would be Invivofectamine™ 3.0 Transfection Reagent, which is a nanoparticle technology that can encapsulate and protect the payload for delivery. It can be used on cells in vitro as well as injected systemically via intravenous route or via direct injection (i.e., muscle, tumors, heart, brain). More information can be found here.

In addition, mRNA can also be synthesized with chemically modified nucleotides to improve stability and further minimize degradation. A positive control mRNA (e.g., GFP mRNA) is very helpful to optimize transfection techniques.

We have not observed differences between how a cell packages an mRNA payload versus a DNA payload for the purpose of delivery. Transfection involves complex formation between a liposome and mRNA, which create lipoplexes that are taken up by the cell via endocytosis. The liposome protects the mRNA during this process and also assists in endosomal escape, which releases the mRNA into the cytoplasm of the cell. The mRNA is immediately available for translation with the ribosome. The mRNA itself should be prepared using an in vitro transcription kit, such as the mMESSAGE mMACHINE® T7 Ultra Transcription Kit, which incorporates a 5’ ARCA cap and a 3’ poly(A) tail so that the mRNA mimics endogenous mRNA.

Lipofectamine™ MessengerMAX™ Transfection Reagent is an animal origin-free transfection reagent, especially formulated for the delivery of mRNA, small RNA (eg.,CRISPR IVT gRNA, siRNA, or miRNA), and short dsDNA or HDR templates (0.5-1 kb). Lipofectamine™ MessengerMAX™ Reagent is an excellent reagent choice for CRISPR-mediated genome editing applications.

Lipofectamine™ MessengerMAX™ Reagent demonstrates low toxicity and high transfection efficiency for all cell types (easy or difficult-to-transfect, primary, and stem cells).

Lipofectamine™ MessengerMAX™ Reagent may be used for both single gRNA delivery or multiplexing (gRNA for multiple targets) purposes with high transfection efficiency when used with GeneArt™ Cas9 mRNA. For a detailed protocol, please refer to the GeneArt™ Cas9 mRNA manual. We recommend using either the Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24732) or Invitrogen™ GeneArt™; Genomic Cleavage Selection Kit (Cat. No. A27663) for mutant analysis.

We recommend using Invitrogen™ Lipofectamine™ RNAiMAX Reagent for the delivery of oligos. We do not recommend using Lipofectamine™ MessengerMAX™ Reagent for the delivery of single-stranded oligonucleotides for genome editing purposes; literature demonstrates that dsDNA HDR templates provide better directed results.

We recommend using Opti-MEM® I Reduced Serum Medium (Cat. No. 31985062).

While Lipofectamine® 3000 or Lipofectamine® 2000 can be used for co-transfection of siRNA with plasmid DNA, Lipofectamine® RNAiMAX cannot be used.  

The PLUS™ Reagent will not enhance transfection efficiency when used in conjunction with Lipofectamine® RNAiMAX.

BLOCK-iT™ Alexa Fluor® Red Fluorescent Control (Cat. No. 14750100) can be used as an indicator of transfection efficiency of siRNA. This dye can be detected using standard filter sets designed for Alexa Fluor® 555, Cy®3, DsRed, Texas Red®, or rhodamine fluorophores.

We recommend using the Lipofectamine™ RNAiMax Transfection Reagent. Please access the protocol here. Both our mirVana inhibitors and Ant-mirs may be used at a final concentration of 5 nM for effective knockdown effects.

Lipofectamine Stem Transfection Reagent is a lipid-based transfection reagent that is specifically designed for stem cells. It is our most versatile, and preferred choice for delivering DNA, mRNA, and Cas9 protein (RNP) into human pluripotent stem cells, as well as neural stem cells (NSCs) and mesenchymal stem cells (MSCs). Lipofectamine 3000 Transfection Reagent can be used to deliver DNA or RNA and is recommended for hard-to-transfect cell lines and workhorse cell lines.

Lipofectamine Stem Transfection Reagent can be used to deliver and co-deliver DNA, mRNA, and Cas9 protein (RNP) into stem cells, including neural stem cells (NSCs) and mesenchymal stem cells (MSCs).

Yes. Most albumin- and serum-supplemnted media do not inhibit transfection with this reagent.

Yes. High transfection efficiency can be achieved in stem cells in suspension with reverse transfection using Lipofectamine Stem Transfection Reagent. Stem cells can easily be transfected at the time of passaging and during re-plating, when they are in suspension. For simple instructions on using reverse transfection with Lipofectamine Stem Transfection Reagent, please take a look at the transfection protocols for pluripotent stem cells, listed under Documents, Product Literature on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product).

Yes. Lipofectamine Stem Transfection Reagent has been tested with StemFlex Medium and Essential 8 Medium on Vitronectin, Geltrex, and Laminin-521 matrices.

We recommend using our feeder-free Essential 8 Medium or StemFlex Medium on Vitronectin, Geltrex or Laminin-521 matrices. Lipofectamine Stem Transfection Reagent also supports other media systems such as mTeSR1 on Geltrex matrix.

We recommend using Lipofectamine RNAiMAX Transfection Reagent for siRNA delivery.

When planning your transfection-based experiments, it is important to optimize the amount of nucleic acid and transfection reagent for each stem cell type being used, so that you may balance optimum transfection efficiency with minimal cytotoxicity.

Proliferating stem cells in culture need room to expand. We recommended plating cells so they will be 30-60% confluent at the time of transfection. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

Yes. Lipofectamine Stem Transfection Reagent is optimized for genome editing in human pluripotent stem cells and neural stem cells. Lipofectamine Stem Transfection Reagent provides efficient transfection of large DNA plasmids such as CRISPR/Cas9 vectors, RNA such as Cas9 mRNA co-transfected with tracr/guide RNA, or Ribonucleoprotein complexes of Cas9 protein with guide RNAs.

Certain components of culture media can be inhibitory to lipid-based transfections. For best results, we recommend using OptiMEM Reduced Serum Medium for preparing Lipofectamine Stem Transfection Reagent -DNA, -mRNA, and -Cas9 RNP transfection complexes.

Yes. Lipofectamine Stem Transfection Reagent can deliver small and large (up to 11 kb) DNA plasmids into stem cells with very low cytotoxicity.

For cells that have aggregated into large colonies, transfection attempts may result in ONLY the outer cells of the colonies being successfully transfected. Transfection in suspension at the time of passaging and re-plating can maximize access of the reagent to all the cells and increase transfection efficiency. We recommend cell culture conditions that promote formation of a monolayer versus clumps of cells. To avoid cell aggregates that form large colonies, gently dissociate colonies to small clumps of 3-5 cells to promote efficient transfection. Follow recommended seeding densities for the cell culture medium and passaging reagents used so that cells are at the recommended starting confluency of 30-60% at the time of transfection and have room to grow for 24-48 hours during transfection.

Yes. Lipofectamine Stem Transfection Reagent can be used to transfect a wide range of stem cell types, while supporting continued proliferation without inducing differentiation. It is our recommended solution for transfecting neural stem cells.

Cell confluency can impact the amount of Lipofectamine Stem Transfection Reagent that is needed for efficient transfection. Stem cells that are near confluency do not transfect well. Plating density should be such that cells are not overgrown 24 hours after transfection. To obtain the highest percent of stem cells transfected, we highly recommend that you dissociate both colony type and monolayer cultures into small clumps of 3-5 cells. If you are growing human pluripotent stem cells in a 24-well plate in Essential 8 Medium on Vitronectin, plate the cells to achieve 30-60% confluency on the day of transfection. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

Yes. Lipofectamine Stem Transfection Reagent can be used to co-deliver multiple payloads, including DNA, mRNA, and Cas9 protein (RNP) into stem cells.

To obtain the highest percent of stem cells transfected with Lipofectamine Stem Transfection Reagent, we highly recommend that you follow cell culture conditions that promote formation of a monolayer versus clumps of cells. If the density of your stem cells is greater than 50% or they are being grown as colonies in other media, more Lipofectamine Stem Transfection Reagent may improve transfection efficiency. For additional details, please refer to the Lipofectamine Stem Transfection Reagent protocol pertinent to your cell type and media on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product), under Documents, Product Literature.

You may perform up to 10 injections of 200 µL each of a 0.1 mg/mL siRNA complex solution (e.g., 20 g mouse, 1 mg (siRNA)/kg (body mass)).

The Invivofectamine™ 3.0 Reagent protocol was validated in-house for delivery to the liver by mouse tail vein injection. However, delivery to the brain and spleen has also been observed by our customers.

Invivofectamine™ 3.0 Reagent-siRNA complexes may be stored up to two weeks at 4 degrees C.

The Invivofectamine™ 3.0 Reagent-siRNA complexes may be used without the 6-fold dilution, in concentrated form. However, the complexes should be either dialyzed or diafiltrated before use.

Invivofectamine™ 3.0 Reagent is an improved formulation with the following increased benefits:

• Lower toxicity
• Higher transfection efficiency with less siRNA; sustained knockdown up to 2 weeks
• Increased stability
• Shorter overall workflow; no dialysis or diafiltration needed

Your siRNA must be at least 2.4 mg/mL in RNase/DNase-free water before complexing. siRNA stock concentrations lower than 2.4 mg/mL will result in low encapsulation efficiencies. We also recommend confirming the siRNA concentration before complexing with Invivofectamine 3.0 Transfection Reagent to verify the correct concentration. After resuspension in RNase/DNase-free water, measure the OD at 260 nm of several serial dilutions of the siRNA stock and calculate the concentration.

To ensure the best results and minimal toxicity, it is very important to determine the weight of each mouse to provide the correct dose and to maintain uniform dosing of multiple animals. The recommended dose for mice is 1.0 mg/kg (intravenous), depending on the potency of your siRNA.

We recommend using Ambion In Vivo siRNA or either of the Silencer or Stealth RNAi in vivo ready siRNAs with Invivofectamine 3.0 Transfection Reagent. BLOCK-iT fluorescent controls may be used to track biodistribution of siRNA. Invivofectamine 3.0 Transfection Reagent offers the following benefits over the discontinued Invivofectamine 2.0 Transfection Reagent:

- Lipid-based delivery for easy-to-use systemic in vivo siRNA delivery to the liver by tail vein injection
- Animal origin-free reagent formulation
- Increased transfection efficiency with reduction in siRNA required per injection
- More stable complexes for sustained knockdown up to two weeks
- Lower cytotoxicity
- Shorter protocol

For more detailed information on the Invivofectamine 3.0 Transfection Reagent please go here (https://www.thermofisher.com/us/en/home/life-science/rnai/introduction-to-in-vivo-rnai/invivofectamine-reagent.html).

Sterile technique is vital to successful completion of in vivo experiments. All materials need to be sterile before injecting the complexed siRNA into animals.

We recommend the use of inbred strains for in vivo experiments, because these strains provide a uniform experimental background in which to determine gene knockdown and downstream effects. In addition, we recommend purchasing animals that are in good health from a qualified vendor. Starting with sick or lethargic mice is likely to result in problems during the experiment

Yes, we recommend that you test at least 3 different siRNA duplexes in vitro at several different concentrations and use the siRNA duplex with the highest potency for your in vivo experiments. When moving from in vitro to in vivo experiments, it is also important to double check the species specificity of the duplex.

Invivofectamine 3.0 Reagent is designed to be delivered slowly and evenly; a 200 µL injection should take between 10 to 20 seconds.

Yes, you may do so by using a 0.22 µm pore size for filter-sterilization. However, our best recommendation is to always keep your procedure sterile from start-to-finish.

Isolating high-quality RNA is critical to the overall success of in vivo experiments. Please visit the Invitrogen RNA Purification page for how to best handle and isolate RNA from in vivo samples.

We recommend that your siRNA is of the highest quality available. In vivo purity siRNA from Invitrogen or highly purified and desalted siRNA is required for good results. Failures in synthesis, excess salts, free nucleotides, and improperly purified siRNA may result in poor performance in vivo.

Invivofectamine 3.0 Reagent is optimized for liver delivery after tail vein injection.

We recommend using housekeeping genes for normalization. The expression levels of housekeeping genes can vary depending on the experimental conditions and/or tissue type. We have obtained the most consistent results by normalizing target gene expression to HMBS and GAPDH.

We recommend starting with a dose of 1.0 mg/kg as a starting point for experiments. This dose corresponds to 200 µL of a 0.1 mg/mL solution injected into a 20 g mouse (i.e., 10 µL/g).

Complexes prepared with Invivofectamine 3.0 Transfection Reagent do not require either diafiltration or dialysis. But if dialysis is needed, any of the Slide-A-Lyzer dialysis systems may be used, as long as the MW cutoff is 50 kDa. At the end of dialysis, you may observe some volume increase. If volume reduction is needed, follow dialysis with a Pierce Protein Concentrator but ensure the final volume is no less than the total undiluted complex volume to avoid binding issues to the membrane. Alternatively, dilute the complex with PBS by 6-fold and directly concentrate until the total volume is back to no less than the starting volume.

Please go to our our dialysis, desalting, and concentration selection guide here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration.html

Complexes prepared with Invivofectamine 3.0 do not require either diafiltration or dialysis. However, if diafiltration is used, any of the Pierce Protein Concentrator PES systems may be used, as long as the MW cutoff is 50 kDa. Keep the final volume to no less than the total undiluted complex volume. Please go to our concentrator selection guide here.

Keep the siRNA concentration at 2.4 mg/mL and follow protocol instructions for complex preparation, keeping the siRNA to Invivofectamine 3.0 Reagent ratio at 1.2:1 (µg:µL).

The Lipofectamine CRISPRMAX Reagent was developed to efficiently deliver the Cas9-gRNA ribonucleoprotein complex. We recommend that you first deliver the donor plasmid with Lipofectamine 3000, then follow with delivering the Cas9-gRNA complex with Lipofectamine CRISPRMAX Reagent for best editing efficiency.

Although the Lipofectamine CRISPRMAX Reagent was developed for the delivery of the Cas9-gRNA ribonucleoprotein, there is potential for other protein complex applications.

The Lipofectamine CRISPRMAX Reagent combined with the proprietary enhancement properties of the Lipofectamine Cas9 Plus Reagent leads to efficient complex formation with the Cas9-gRNA ribonucleoprotein, for best delivery to the nucleus, helping to ensure high gene editing frequency for a wide range of cell types.

Please see the Lipofectamine CRISPRMAX Reagent manual (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine_crisprmax_man.pdf) for protocol details or contact techsupport@thermofisher.com.

Lipofectamine™ 3000 Reagent may be used with Jurkat cells but the highest transfection efficiency (~98%) can be achieved using the Neon™ Transfection System. 

We recommend using the Lipofectamine™ 3000 Reagent for transfection of endothelial cells.