I wish to label SHS5Y cells with a fluorescent lipid dye so that I can measure the diffusion coefficient of the dye in a plasma membrane. I have to use Rhodamine-DOPE or another red dye. Does anyone have any suggestions? I tried incubating with Rhodamine-DOPE in a small amount with cells for 30 minutes at room temperature and washed out the unbound dye, but it gave patchy labeling. Are there any specific methods for labeling cells?
Do you wish to bind a fluorescent dye on the surface or cytoplasm or nucleus of the cells? Each of these experiments must have different procedure. Incubation on the room temperature could be used only for adhesion of the dye on the surface of cell membranes, but even for this experiment cell environment must be modified, including the surrounding atmosphere and temperature.
I want to measure the lateral diffusion of the dye in the plasma membrane,,so only the surface. Hence I want to use a fluorescent lipid which upon addition to the cell media should fuse with the cell membranes. I do not mind if the fluorescent lipid enters the cell, because i think they are weakly fluorescent in water..
My guess is that the lipids are not properly dispersed. Rhodamine DOPE will have a strong tendency to form vesicles or precipitates which may cause patchy labeling. Adding a cell tolerant detergent like pluronic may help disperse aggregates and help more uniform labeling.
Maybe you can first isolate cell membranes and than develop procedure for labelling according suggestion of Jeffrey.
Zlatko
Use NBD-stearate available from Avanti. With the proper confocal microscope system, you can do this experiment with a simple FRAP routinet. We reported data using this experiment in Am. J. Phys years ago. You can do the experiment either for membrane diffusion coefficient or set it up to do cytoplasmic diffusion coefficient.
If you are looking at extracellular membrane, I would still use an NBD-labeled lipid and one with a choline headgroup as opposed to ethanolamine labeled head group. So a NBD-PtdCho (NBD-PC) would work and should be available. I would check Avanti Polar Lipids first.
I can try with the NBD-PC which we have avaialble. But i wanted a red label rather than a green one. Will DiI work? I will try out with NBD and let you know! Thanks Eric!
I have labelled the cell membrane with DiI. I use a 1mg/ml solution in ethanol, add 1ul of this per 1ml of medium and incubate for 10-20min. You need to test the time, when you incubate too long, it penetrates inside and colours the internal membranes as well.
The labelling is uniform, except for some structures in the basal membrane which you can also see when labeling with a protein (e.g. GPI anchor transfection).
I think your patchy staining is endocytosed membrane. 30mins at RT is quite a long time for the incubation. I dilute rhoPE to around 1-3 ug/ml in PBS, put it on the cells for 10mins on ice, and get nicely uniform, very bright staining of the PM. A couple of notes of caution though... rhoPE is closer to orange than red, so will easily leak into your green channel. Also, it has a tendency to photo-oxidize, which can have lots of unwanted effects on the membranes you're measuring. So minimize the excitation light intensity as much as possible.
i prepared lipid bilayer membrane using vesicles with lipids: DPPE and DPPS with ratio 2:1. How can i attach dye with it? Which type ofdye is most suitable? I will estimate diffusion coefficient of bilayer membrane deposited on solid state nanopore biosensor.
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