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 info@ibidi.de 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 products are 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-Reagents?

First, add Chloroform or an alcohol to one Fuse-It vial with lyophilized lipids (e.g., Fuse-It-P [Cat. No. 60220, 60221, 60222, or 60223] or Fuse-It-L [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 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 info@ibidi.de 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.