Collot, M.; Fam, T. K.; Ashokkumar, P.; Faklaris, O.; Galli, T.; Danglot, L.; Klymchenko, A. S. Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues. J. Am. Chem. Soc. 2018, 140 (16), 5401–5411. https://doi.org/10.1021/jacs.7b12817
Ponsot, F.; Shen, W.; Ashokkumar, P.; Audinat, E.; Klymchenko, A. S.; Collot, M. PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience. ACS Sens. 2017. https://doi.org/10.1021/acssensors.7b00665
Versatility: compatible with liposomes, exosomes, apoptotic bodies, living and fixed cells, spheroids, organoids, tissues, etc.
5 colors: emission from 500 nm to 700 nm.
Bright and photostable: used at low concentrations of 20 to 200 nM for cells in culture. Suitable for long-term imaging
Fast staining: 2-15 min
Reduced tocxicity: The brightness of MemBright allows it to be used at low concentrations (as low as 10 nM) with low excitation power.
Short-term preservation: at + 4 ° C protected from light
Long-term storage: at -20 ° C protected from light
Protocol of use
For adherent cells:
Before staining, prepare a solution of MemBright in your visualization medium such as PBS, HBSS, MEM, Opti-MEM, Krebs, etc. (Caution: do not use medium with serum) with a final concentration ranging from 10 nM to 200 nM.
Remove the sample from its support, wash the sample twice (for example with PBS) and cover your cells with the freshly prepared MemBright solution (do not store this solution for more than a minute without cells).
No need to wash the cells of the dye, visualize directly.
Alternatively, it is also possible to add a small volume of a diluted solution of MemBright (4 to 40 μM) directly into the middle of your sample, then mix gently.
All MemBright stock solutions are prepared in DMSO.
Which medium should I use for my experiments ? These probes were designed to operate in serum-free conditions because the serum would desorb or evacuate the probes from the membrane.Long-term experiments in serum-free conditions are possible if you use a specific medium.Please consult the following links: https://bioscience.lonza.com/lonza_bs/CH/en/serum-free-cell-culture https://www.thermofisher.com/fr/fr/home/life-science/cell-culture/mammalian-cell-culture/serum-free-media.html
The recommended media is also BSS, PBS, MDEM, Oti-MEM, etc.
Is Lipilight compatible with the fixation technique ? The best technique is to first stain the cells with Lipilight and then fix the cells using the corresponding medium.After fixation, the staining of the membrane may slightly decrease, but must remain strong. Permeabilization is not recommended because it creates pores in the plasma membrane that lead to internalization of the probe.
How should I dilute my stock ? The original stock solution is provided in DMSO in which the probe is fully soluble.If you dilute your stock solution (in DMSO) in an aqueous medium (PBS, DMEM, etc.), you must immediately transfer this solution to the cells, otherwise aggregates would tend to form with a decrease in the effectiveness of the solution.coloring You can also dilute the stock solution in DMSO.In this case, be careful not to add too much DMSO to the cells (<0.5%) to avoid cytotoxic effects.Although direct addition to cells gives excellent results, it is possible that you have cytotoxicity problems because the local concentration of DMSO / probe at the point of addition may be too high.
How long will the staining remain in the plasma membrane ? Depending on the Lipilight used, the probe remains approximately 30 to 45 minutes at the level of the plasma membrane, then begins to be internalized and the endosomes can be visualized.The cells can then be imaged for several hours. Lipilight 488 is definitely the best for long-term imaging. Warning :The phototoxicity of membrane probes is frequently related to photooxidation of lipids.In this case, you can try an anti-oxidant, astaxanthin.It had been previously reported that this kind of problem was solved (Gonzalez & Tsien, Chemistry 8 Biology 1997, 4, 269).
Is there a substrate or material to avoid when imaging my probes ? You must avoid any hydrophobic substrate or detergent.The addition of Lipilight to these samples will induce a significant background noise.Nevertheless, you can always try to pre-color with Lipilight, then add it to your hydrophobic substrate after cleaning the staining medium.
Can I get an image of organoids or systems much larger than separate cells ? Collot et al.Bioconjugate chem 2019 has shown that effective staining is proportional to the number of lipids to be stained.Thus, the typical concentrations for organo-color are more in the micro-molar range.We are working on a stock with higher concentrations for these types of applications.
What are the differences in color throughout the Lipilight range ? The design of different colors has led to structural variations and therefore to differences in solubility, aggregation, and kinetics of staining and efficiency as a function of the samples.In addition, Lipilight-488 is based on BODIPY and is therefore globally neutrally charged whereas the other cyanine-based probes are globally positively charged, which may also explain some differences in staining efficiency.You can refer to additional materials from Collot et al.Cell Chem Biol, 2019.