FAQ

FAQ general

 

What can be fused with Fuse-It?

Fuse-It works with a number of mammalian cell lines, primary cells, several types of tissue, as well as artificial membranes e.g. small or giant unilamellar vesicles (SUVs or GUVs).

How stable is Fuse-It?

The fluorescent marker of the cell membrane is present in cells for at least 24 hours. In cells with a high metabolism rate the fluorescent dye is more rapidly internalized than in cells with a lower metabolism rate. Even after fixation and permeabilization, the color is still present, which enables additional staining of antibodies.

Where does Fuse-It stain?

Fuse-It stains the unordered phase (fluid-crystalline phase) of the lipid membrane. A selective marker of the ordered phase is possible on request. Please contact info@beniag.com or info@ibidi.de for more information.

What do I have to consider during the sonication of Fuse-It?

At a frequency of around 35 kHz the ultrasonic bath should have a power of 100-800 W. During the whole sonication, the Fuse-It solution should not exceed a temperature of 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 system is cooled when using a probe.

How do I store Fuse-It?

All Fuse-It products are sent at 4°C and must be stored upright at -20°C upon arrival (except for tester – storage at 4°C). The Fuse-It solution shouldn’t be aliquoted and stored in test-tubes made out of plastic. However, a transfer of the required amount for immediate use is possible.

How highly should Fuse-It be diluted?

For an efficient fusion a dilution of 1:50 to 1:100 is adequate.

What size do sonicated Fuse-It vesicles have?

After a sonication of 10 minutes an approximate size of 300 nm is reached.

What can I do when Fuse-It does not work well on my cells?

Fuse-It is supposed to work approximately the same for lots of types of cells. A special fusion system was developed for difficult fusogenic cells. Please contact info@beniag.com or info@ibidi.de for further information.

 

FAQ Fuse-It tester

 

What does the Fuse-It tester do?

The Fuse-It tester was developed to try the fusion of your cells of interest. The Fuse-It tester consists of the same base components as the standard Fuse-It products but is provided at no cost for trial purposes. A successful fusion can show immediately how compatible Fuse-It is with your cells. The Fuse-It tester contains liposomes with red fluorescent, amphipathic molecules in its membrane and green fluorescent beads (50 nm) in its lumen. Both molecules are transported into the cells. As a result, cells with a red fluorescent cell membrane and green fluorescent beads in the cytosol are obtained.

Why is it not possible to transport proteins, lipids or beads in the Fuse-It tester?

The Fuse-It tester is made up of finished liposomes, which wrap the supplied particles. For a transport of proteins and lipids, the liposomes would have to be built of multilayered, lyophilized layers of lipids (Fuse-It-P or Fuse-It-L), together with the chosen molecule. For the transport of beads and nanoparticles, the specifically matched Fuse-It-Beads are sonicated with the particles. This leads to the enclosure of the particles by the liposomes’ lumen, or, for bigger particles, an enclosure by the liposome itself.

Why do I see the green fluorescent beads outside the cells even after washing?

The beads consist of polystyrol latex and therefore are seen as a foreign body by the cell, which leads to their discharge from the cell. This is why the examination of the fusions’ efficiency should be done directly after the fusion if possible.

Why is the green fluorescent signal so weak compared to the red fluorescent signal?

The beads for staining the cytosol only have a small number of fluorophores. Also it is necessary to mention that the amount of beads per cell is limited. Furthermore the beads are seen as a foreign body by the cell and so they are actively expelled over time.

 

FAQ Fuse-It-P

 

How big can my protein be when using Fuse-It-P to transport it into cells?

Currently, the biggest protein that was transported by fusion was phycoerythin with around 250 kDa.

How much of my protein arrives in my cells as a result of the fusion of Fuse-It-P?

With a starting concentration of 0.8 mg/ml phycoerythin it was shown that an intracellular protein concentration of 1-10 µg/ml can be reached.

Which condition does the buffer have to have to combine Fuse-It-P and my protein?

Lower molecular buffers (i.e. 20 mM HEPES) are most suitable with a pH of 7-8. To improve the solubility, the buffer’s molarity can be increased up to 150 mM.

Which additions of the buffer can cause trouble during the creation of Fuse-It-P-Liposomes?

Generally, a glycerol concentration of over 0.1% as well as the presence of many frequently used surfactants can inhibit the creation of Fuse-It-P-Liposomes. If necessary the protein should preferably be present in a high concentration so that the glycerin 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. Sucrose is not a problem for the creation of Fuse-It-P-liposomes. The total protein concentration needs to be under 1 mg/ml. This is to be observed by the use of BSA or other protein additions of the buffer.

 How stable are my proteins during the fusion?

In the liposomes, the proteins are as stable as they are when stored in the buffer at -20°C. With antibodies it was shown that incorporated proteins were functional even after vortexing for 10 minutes and a 20 minute ultrasonic bath.

Why does the lyophilized Fuse-It-P not dissolve completely after adding my protein solution?

A high protein concentration can decrease the solubility. If this happens, resuspending with a pipette (10-20 x) can help and vortexing after opening the lid only a few millimeters.

What has to be considered when working with highly charged proteins?

Proteins with a total charge from 10 to -30 mV can be transferred without further ado. The concentration of the positive charged ions Na, Mg and Mn can be increased to no more than 150 mM in total, when working with more highly charged proteins and a local charge density.

 

FAQ Fuse-It Beads

 

What types of beads and nano particles can be transferred to cells with the help of Fuse-It-Beads?

This is possible for all solid and partly solid particles. The small particle will be enclosed in the vesicle’s lumen. Bigger particles are wrapped by the Fuse-It-Beads solution. Week positive or negative surface charges do not cause any problems.

How big are my particles allowed to be?

Particles with a diameter of 5 µm can be transported into the cells by Fuse-It-Beads.

 

FAQ Fuse-It-B

 

What is the percentage of biotinynated lipids in Fuse-It-B?

The percentage is 5 mol%.

Why do I not see a consistent signal on the cell surface after the fusion and staining of the biotin molecules?

In Fuse-it-B, biotin is coupled to a phospholipid which will be inserted into the cellular plasma membrane via membrane fusion. After insertion, the foreign molecules will be partially discarded from the membrane. Due to this natural process of molecular digestion the biotin signals are locally altered.

 

FAQ Fuse-It-mRNA

 

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.

 

FAQ Customize Fuse-It

 

We are happy to adapt Fuse-It to your individual needs. Please contact info@beniag.com or info@ibidi.de for more information.

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