Which types of membranes can be fused with the Fuse-It products?
Fuse-It works with a number of mammalian cell lines, primary cells, several types of tissue, artificial membranes, and giant unilamellar vesicles (GUV). Please contact [email protected] for specific information.
How stable is Fuse-It after fusion?
The fluorescent staining of the cell membrane is usually stable for 24 hours. In cells with a high lipid metabolism, the dye-coupled lipids are being internalized over time and might show internal staining after some hours, in addition to the membrane staining. However, in many cell types, the staining can be used even after one week for the identification of fused cells with microscopy or flow cytometry. Even after fixation and permeabilization with non-alcoholic reagents, the staining is still present, and allows for additional stainings with antibodies.
What do I have to consider during the sonication of Fuse-It?
At a frequency of 30 – 40 kHz, the ultrasonic bath should have a power of 100 – 800 W. During the whole sonication, the Fuse-It solutions’ temperature should not exceed 25°C. Therefore, the ultrasonic bath might have to be cooled down with ice, if necessary. The use of an ultrasonic probe is not recommended, as temperatures in this area might get too high. In case no other equipment is available, it is essential that the temperature be cooled down.
How do I store Fuse-It?
All Fuse-It productsare sent on cool packs and must be stored upright at -20°C. The Fuse-It solution should not be aliquoted into tubes made out of plastic for long-term storage. However, it is possible to transfer the required amount into plastic tubes for immediate use.
How should Fuse-It be diluted?
For an efficient fusion, a dilution of 1:50 to 1:100 is adequate.
How can I make aliquots of the lyophilized Fuse-It-L?
First, add Chloroform or an alcohol to one Fuse-It-L vial with lyophilized lipids (Cat. No. 60210, 60211, 60212, or 60213). The final lipid stock concentration should be 3 mM. Next, vortex until the solution is homogeneous (if necessary, mix by pipetting), then transfer the appropriate lipid stock volume into glass vials. The minimum aliquot size should be 10 µl. Afterwards, evaporate the solvent entirely under vacuum or reduced pressure for at least 30 min. The aliquots are now ready to use for your fusion experiments. Store the lyophilized Fuse-It-L aliquots at -20°C.
What size do sonicated Fuse-It vesicles have?
After a sonication of 10 minutes, Fuse-It vesicles show a size distribution of 300 to 380 nm.
What can I do when Fuse-It does not work well on my cells?
Fuse-It is shown to be highly efficient on many cell types. However, if you are using cells that are difficult to fuse (e.g., endothelial cells), we can offer you a special formulation that was developed for low fusogenic cells. Please contact [email protected] for more information.
I do not have an ultra-sonication bath. Are these steps really necessary?
Yes. Ultra-sonication prevents liposomal aggregation and, therefore, ensures homogenous and efficient fusion. Most standard sonication baths will be sufficient for preventing aggregation.
Which receptors on the cell surface are involved in the fusion process?
In contrast to any other nucleic acid transfer methods (except for electroporation), fusion is exclusively driven by physicochemical-attractive interactions between the plasma membrane and the lipid bilayer of fusogenic liposomes. Electrostatic interactions are of main importance because fusion will even occur on artificial lipid bilayers without any incorporated protein. This receptor-independent interplay allows for the use of Fuse-It-mRNA with almost every mammalian cell type.
How do you guarantee that the mechanism is really fusion and not endocytosis?
A few experiments have already been performed, and all of them have proved the efficient fusion of fusogenic liposomes with cellular plasma membranes. Examples are: the use of molecules that inhibit endocytosis, dyes that change their emission spectrum, depending on whether they are incorporated in vesicles or freely diffused within the plasma membrane (Braun et al. 2016, Cytometry, 89:301-308), biomimetic systems that use giant unilamellar vesicles, or the diffusion analysis of transferred molecules within the plasma membrane by FCS. In addition, you can easily visualize the membrane fusion process and control the fusion in your own sample by following an IR dye in fluorescence microscopy that is incorporated into the Fuse-It-mRNA fusogenic liposomes (Ex. 700 nm, Em.780 nm). Homogenously distributed fluorescence staining of the plasma membrane indicates successful membrane fusion.
Which Fuse-It reagent would you use to incorporate an ATTO Dye, for example, and SNAP Tag to target a certain cellular structure, such as for super resolution applications?
It depends on whether your dye is soluble in water or an organic solvent. For dyes bound to amphipathic molecules, we strongly recommend Fuse-It-L. For water-soluble dyes or fluorescently-labeled substrates (e.g., SNAP, HALO or CLIP-systems), you can use either Fuse-It-P or Fuse-It-L, as well. Just follow the protocol and swell (for Fuse-It-L) the fusogenic liposomes directly in the solution of molecules or dyes of interest.
How much mRNA is needed to adequately express proteins?
For best results when using the µ-Dish 35mm, high, use the following ratios: 1 µg mRNA : 2 µl NB and 2.5 µl FS : 250 µl total volume. The amounts for other formats are listed in the Fuse-It-mRNA instructions. The ratios of reagents for formats other than those listed will require optimization.
Can I transfer two mRNAs at the same time using Fuse-It-mRNA?
Yes. When using a µ-Dish 35mm, high, use a total of 1 µg mRNA. For best results, maintain the following ratios: 1 µg mRNA : 2 µl NB and 2.5 µl FS : 250 µl total volume.
Can I use Fuse-It-mRNA for the transfer of siRNA?
No. The Fuse-It-mRNA protocol is optimized for the transfer of mRNA. We recommend using the Torpedo siRNA (Cat. No. 60620, 60621, and 60622) for the transfection of mammalian cells with siRNA and miRNA.
What is the difference between the two components in the Fuse-It-mRNA?
The Neutralization Buffer (NB) compensates for the negative charge of the mRNA. The Fusogenic Solution (FS) is a lipid formulation that forms a vesicle around the neutralized mRNA. The liposomal carriers are able to attach and fuse with cell membranes.
I am already using other Fuse-It products. Can I use the same cell-dependent incubation time for the Fuse-It-mRNA protocol?
No, the standard protocol is optimized for mRNA transfer; however, the incubation time must be optimized for each cell type. Please refer to the Fuse-It-mRNA product page for a list of cells that have been tested with Fuse-It-mRNA and the corresponding incubation times.
What should I do if I cannot find my cell type in the list of cells tested with Fuse-It-mRNA?
For first-time use of Fuse-It-mRNA, we recommend following the standard protocol. For optimization, you can adjust the mRNA concentration, the incubation time, the dilution medium, or the cell confluence.
What should I do if I do not see any expression after fusion?
An IR-dye has been incorporated into the fusogenic vesicles for transfer verification. The emission of the IR-dye at 780 nm will allow you to check if the fusion process was successful.
Furthermore, the liposomal carriers can interact with plastic surfaces. To minimize carrier loss, make sure you have good cell attachment and high cell confluence. Coatings, such as fibronectin, collagen, or poly-lysine, may help optimize cell attachment and confluence.
What should I do if my cells are fused but my mRNA is not expressed?
If cells are fused and no expression can be detected within 24 hours, check the mRNA quality. Also check the ratios. For example, when using the µ-Dish 35mm, high, use 1 µg mRNA : 2 µl NB and 2.5 µl FS : 250 µl total volume.
Is Fuse-It-mRNA toxic for my cells?
No. A small amount of liposomal carrier is used for mRNA transfer, and chemical compounds for endosomal release are not necessary.
Is it okay to sonicate nucleic acids?
Sonication wavelengths in standard sonication baths are much longer than the length of your mRNA - no matter how long your mRNA might be. Therefore, sonication homogenizes liposomal size distribution but does not harm your mRNA.
Where do I get mRNA?
You can purchase high-quality mRNA from companies such as Eurofins GmbH, TriLink BioTechnologies, Inc., Allele Biotechnology and Pharmaceuticals, Inc., or AMS Biotechnology Ltd.
Alternatively, you can synthesize your own mRNA with commercially available kits. For example, you can use the “MessageMAX™T7 ARCA-Capped Message Transcription Kit” from CELLSCRIPT™, the “HiScribe™ T7 RNA Synthesis Kits” from New England BioLabs GmbH, or the “mMESSAGE mMACHINE™ Kits” from Thermo Fisher Scientific Inc.
Which mRNA qualities should I be concerned about?
The mRNA should meet the requirements of your application. The more stable the mRNA is, the longer the protein will be expressed in the cells. Best results are achieved with functionally capped and polyadenylated mRNAs, but it may not be necessary depending on the experiment. Other mRNA modifications also do not affect the fusion process.
The purity and the concentration can be measured using a spectrophotometer: The 260/280-ratio should be approximately 2.0 (pure RNA), and the 260/230 ratio should be ~2.0-2.2. The concentration needed is 0.5-2 μg/μl in buffer (e.g., TE-buffer).
Can I use Fuse-It-mRNA to produce iPS cells?
Yes, you can. A couple of companies offer all of the necessary, mRNA-based transcription factors (e.g., Oct-5, Soc2, or Nanog). Incorporating these factors as an mRNA mixture will generate iPS cells after approximately 14 days. Repetitive incorporation by fusion (e.g., every third day) might be necessary, depending on mRNA stability.
Is Fuse-It-mRNA compatible with the CRISPR-Cas technology?
A few companies offer purely mRNA-based CRISPR-Cas systems. These systems can be effectively used in combination with Fuse-It-mRNA. In addition, whenever necessary, Fuse-It-mRNA can be combined with classical lipofection. Since fusion does not cause any cell stress, immediately after sgRNA incorporation by fusion, for example, plasmid encoded Cas9 could be incorporated by lipofection.
Has Fuse-It-mRNA been characterized for use in vivo in mice for delivery in, for example, muscle or skin?
No. In vivo experiments have not yet been performed using Fuse-It-mRNA. However, based on another Fuse-It product, namely Fuse-It-tissue, we know that fusogenic liposomes efficiently fuse various layers of 3-dimensional tissue slices. Therefore, the application of Fuse-It-mRNA to tissue surfaces should be possible, and result in good penetration depths.
Is the cell seeding density per well the same for all cell lines?
In principle, you can use Fuse-It-mRNA with any cell density. However, we recommend confluency in the range of 70 to 80%. This is because fusogenic liposomes simply sink to the cell culture bottom, due to their slightly higher density compared to the medium. The more cells you have on the surface, the more liposomes will find their way to the plasma membranes.
How stable are cells after the fusion step?
Fusion itself is a very mild method. As long as you are following the protocol, cells remain as stable as they would without fusion, and can be used immediately for further analysis.
How long are cells expressing the transgene after fusion? Is it up to 10 days, depending on the cell line?
The stability of incorporated, mRNA-based proteins depends on several aspects that are independent of the fusion itself. Most importantly, every mRNA, as well as the resulting protein, has its specific, natural stability. Proteins can be stable over days or just for a few minutes. Therefore, if you are interested in stable protein levels lasting over several days, make sure to fuse mRNA molecules of high quality. This includes 5’- and 3’-untranslated regions, a long polyA-tail, as well as a 5’-cap. The 5’- and 3’-untranslated regions do not necessarily have to belong to the open reading frame of interest, but can originate from any stable mRNA. When using such mRNAs, analysis times are at least comparable to classical transient plasmid incorporation. It is possible to achieve stable protein levels over three days for highly proliferative cells, and up to 10 days for matured cell types.
Why is Fuse-It-P delivered in lyophilized form?
Lyophilization allows for the formation of fusogenic liposomes in buffer conditions, which are optimal for your protein of interest. As long as your protein/peptide buffer is free of other stabilizing proteins (e.g. BSA), and has an osmolarity of not more than a maximum of 150 mOsm (ideal are osmolarities of up to 50 mOsm), any buffer can be used. Thus, fusogenic complexes form directly from the lyophilisate in your protein/peptide solution. This will result in optimal incorporation of the proteins/peptides into the lumen of the resulting liposomes.
How long is Fuse-It-P stable after loading it with protein?
The incorporated proteins/peptides might interfere with liposomal function or stability over time. Therefore, we recommend the direct use of protein-loaded liposomes and the generation of only the specific amount of fusogenic mixture needed. If absolutely necessary, store the remaining loaded liposomes over night at the temperature, which is recommended for your protein. If your experimental setup requires less Fuse-It-P than included in the kit, we recommend aliquotation. Please refer to “How can I make aliquots of the lyophilized Fuse-It-Reagents?“.
How can I make aliquots of the lyophilized Fuse-It-P?
First, add Chloroform or an alcohol to one Fuse-It-P vial with lyophilized lipids. The final lipid stock concentration should be 3 mM. Next, vortex until the solution is homogeneous (if necessary, mix by pipetting), then transfer the appropriate lipid stock volume into glass vials. The minimum aliquot size should be 10 µl. Afterwards, evaporate the solvent entirely under vacuum or reduced pressure for at least 30 min. The aliquots are now ready to use for your fusion experiments. Store the lyophilized Fuse-It-P aliquots at -20°C. Fuse-It-P fusogenic liposomes (without incorporated proteins/peptides) are stable for months in indicated buffers. However, we recommend immediate lyophilization.
What is the biggest protein size possible that can be transported into the cells by Fuse-It-P?
Until now, the biggest protein to be transported by Fuse-It-P was phycoerythrin, with around 250 kDa.
How much of my protein will arrive into the cells through the fusion of Fuse-It-P?
With a starting concentration of 0.8 mg/ml phycoerythrin, it has been shown that an intracellular protein concentration of 1-10 µg/ml can be reached.
Which buffer condition of my protein of interest do I need in order to use it with Fuse-It-P?
Lower molecular buffers (20 mM HEPES) are the most suitable with a pH of 7-8. To improve the solubility, the buffer’s molarity can be increased to 150 mM.
Which buffer additives may have adverse effects during the creation of Fuse-It-P-liposomes?
A glycerol concentration of over 0.1% may inhibit the creation of Fuse-It-P-liposomes. If glycerol is necessary in the protein solution, the protein should preferably be present in a high concentration, so that the glycerol concentration can be diluted to a maximum of 0.1% before the creation of the liposomes. If this is not possible, the buffer must be exchanged. In addition, sucrose is not a problem for the creation of Fuse-It-P-liposomes. The total protein concentration must be below 1 mg/ml. This should be considered when BSA or other protein additions are used in the buffer.
How stable are my proteins of interest during the fusion process?
If they were incorporated into the vesicles with their optimal buffer, the proteins are as stable in the liposomes as they are when stored in buffer at -20°C. With antibodies, it was shown that they were functional even after vortexing for 10 minutes and being in a 20-minute ultrasonic bath.
Why did the lyophilized Fuse-It-P not dissolve completely after adding my protein solution?
A high protein concentration may have decreased the solubility. If this is the case, resuspend the solution with a pipette (10-20 times), or vortex it after opening the lid only a few millimeters and see if that solves the issue.
What must be considered when working with highly charged proteins?
Proteins with a total charge of up to -20 mV can be transferred without any problem. However, the concentration of the positive charged ions Na, Mg, and Mn should not exceed 150 mM when working with higher charged proteins and a local charge density.
What is the approximate efficiency for loading small peptides, for example 15 amino acids in length?
It is very difficult to provide a universally valid answer to this question since proteins and peptides are chemically the most heterogeneous molecule group you can find in cells. Depending on the amino acid composition, they can differ in many characteristics (e.g., size, conformation, hydrophobicity, or surface charge). Since the incorporation of molecules via fusion strongly depends on electrostatic interactions between the molecule of interest and the fusogenic liposomes, positive peptide or protein surface charges hamper efficient transfer. Fortunately, most proteins and peptides have an overall negative or neutral surface charge. This allows for the efficient transfer of almost every peptide or protein. In order to calculate the surface charge of peptides and proteins, ibidi provides an application note (coming soon) that will help you to find the best transfer conditions in just a few steps. However, please keep in mind that transferred peptide/protein amounts will reach a concentration in the µM range. This is ideal for preventing overexpression artifacts or for labeling specific structures within the cell, but might be too low to visualize highly abundant cell structures that you might expect when using immunocytochemistry staining.