Vybrant® Lipid Raft Labeling Kits From Molecular Probes (Invitrogen)

Cholera toxin B subunit has been extensively used to stain membrane clusters or rafts. The cholera toxin B subunit targets the ganglioside M1 which is a specific marker of membrane rafts. Most of the studies were performed on T cells to study TCR signal transduction as a pre-signalosome for immune synapse formation. Molecular Probes offer this probe conjugated to Alexa-488 (FITC channel), -555 (PE channel) and -594 (Texas Red channel).

The kit consist of all the reagents needed to perform your staining including the fluorescent probe, the buffer, serum and anti-cholera toxin antibody. You can titrate the dye in order to perform up to 100 stainings. The staining procedure takes about 1 hour. You will need to make aliquots because the dye is still very sensitive to freezing/thawing. You can extensively wash your samples and you will see nearly no background. The staining is performed at 4ºC and this allows you to stop your stimulation while you are doing the staining. The combination of the Vybrant® Kit with other stains is possible and will provide very nice overlays; it is also useful for tracking the association of signaling molecules to membrane rafts during stimulation. It is of note that with flow-cytometry there is much less overlap between dyes because of compensation. This is the reason why, when performing fluorescence microscopy, one should be very careful with the results. I have also used this kit for flow-cytometry determination of GM1 expression; detection of GM1 expression is faster (but less sensitive) than doing it by chromatography.

I have been using this kit to stain T-cells during activation and I am entirely satisfied of the product. I will not change for another, but Molecular Probes needs to increase the variety of membrane raft markers. So far, the cholera toxin B subunit has been the preferred marker; however, there are more and more markers which we have to take into account. Recently, it was shown that membrane rafts are not exclusively GM1+. Using the Vybrant® Kit on melanoma cell lines, I was able to clearly distinguish between GM1+ and flotillin-1+ rafts. Also, the GM3 was shown to partition differently than GM1 during migration or cellular activation.

Finally, this product is the best on the marker so far, very easy to handle and the ratio price/quality is good. Try rafting but only with Vybrant®!