Hello!

I need help! I want to do the localization of lipid rafts. The method look simple but i did that two times and it did'nt work. I use a B subunit of cholera toxin conjugated to AlexaFluor 488 and a confocal microscope. There are the two methods that I tried :

After T cell isolation :

First :

Wash the cells (1 000 000 cells) in PBS 1X and at the end, resuspend the cells in about 10 ul of PBS 1X.
Put the cells on a microscope slide (previously washed with ethanol) and let dry the cells on the the slide for 15 minutes. After do the faxation with 2% paraformaldehyde (in PBS 1X) for 5 minutes. Wash 3 times in PBS 1X.

Do the staining as follwing : Prepare the cholera toxin 1:400 in PBS 1X and put on the cells (20 ul), Incubation at room temperature for 1 hour in a dark and humid environnement. Wash 3 times in PBS with a little agitation and them cover with a drop^of mounting medium (ProLong with Dapi ou glycerol 80%). Add a cover slip and seal...

Second protocol :

Wash the cells (1 000 000 cells) in PBS 1X and at the end, resuspend the cells in 100 ul of PBS 1X.
Cells are treated with the cholera toxin (1:100) in eppendorf tubes for 15 minutes at 4 degrees. Wash the cells 3 times in PBS 1X and resuspend in 10 ul of PBS 1X, put on a microscope slide and cover with a drop of glycerol 80% and a coverslip.

I want to reproduce the experiment like this publication (Acute in vivo elevation of intravascular triacylglycerol lipolysis impairs peripheral T cell activation in humans. PMID: 16280424) and see the localisation of rafts on the T cells.

Could any of you help me? This is suppose to be a very simple technique...

Thank you!

Brassap

hi Brassap,

I have tried many protocols to detect lipid rafts on cell lines, maybe the following tips will be usefull to your experiments...

-do fixation after CTB staining: staining after fixation is very low, maybe due to modification in GM1 ganglioside structure
-do staining in PBS at room temperature or at 37°C: 4°C staining inhibits greatly staining, certainly due to membrane reorganization of the rafts or cytoskeleton activity under the membrane...
-CTB dilution 1:100 or 1:200 max: difficult to observe correct staining at higher dilution (as seen by cytometry)
-for microscpy, just wash the cells in PBS, stain with CTB for 30 min, wash twice in PBS, fix in PFA 2% for 15 min (room temperature), wash twice, resuspend in fluorescent mounting medium and put between slide and coverslipe; store like this at 4°C for some days
-I have observed an increase in staining intensity after activation of the cells: prepare a control with T cells activated with PMA/iono

hpe it will be usefull
bye
Seb

hi Brassap,

I have tried many protocols to detect lipid rafts on cell lines, maybe the following tips will be usefull to your experiments...

-do fixation after CTB staining: staining after fixation is very low, maybe due to modification in GM1 ganglioside structure
-do staining in PBS at room temperature or at 37°C: 4°C staining inhibits greatly staining, certainly due to membrane reorganization of the rafts or cytoskeleton activity under the membrane...
-CTB dilution 1:100 or 1:200 max: difficult to observe correct staining at higher dilution (as seen by cytometry)
-for microscpy, just wash the cells in PBS, stain with CTB for 30 min, wash twice in PBS, fix in PFA 2% for 15 min (room temperature), wash twice, resuspend in fluorescent mounting medium and put between slide and coverslipe; store like this at 4°C for some days
-I have observed an increase in staining intensity after activation of the cells: prepare a control with T cells activated with PMA/iono

hpe it will be usefull
bye
Seb

you may know or even work with Molecular Probes/Invitrogen Vybrant lipid raft labeling kit:

http://probes.invitrogen.com/media/pis/mp34403.pdf

there you will find technical advices

thanks a lot The Bearer,

it is an interesting alternative method for staining of lipid rafts
now, I wonder if crosslinking of CT-B during the second step would not lead to artifactual patching, even if incubated at 4°C...
nervetheless, MP also recommends fixation after staining

Sebatien

Thank you again to you! I will try what you suggested to me!

Thanks!

Brassap