Hello everyone,
I am measuring glucose uptake into my cells by using NBDG and flow cytometry.
Normally this works perfectly fine, but now I got some problems.
When I fixed and permeabilised my cells for the analysis of intracellular proteins after the incubation with NBDG I lost the signal. I guess there might be problems with the stability of the NBDG molecule or it`s leaking out?
Does somebody have experience with that or know some trick to fix the problem?
Thank you in advance,
Katharina
I believe 2-NBDG can survive fixation, but you will lose the signal upon permeabilisation. Maybe try a gentler permeabilisation method?
Permeabilization results in temporary pores in the cell membrane in order to allow large molecules (e.g. fluorescent labeled antibodies) to enter the cell. Those pores are by far large enough to allow your NBDG molecule (which is relatively small) to leak out. My guess is that is why you lose the signal.
You could try to keep your samples on ice after the uptake and throughout the fixation/permeabilization steps. However, even if it helps, i don't think this will accurately reflect the level of glucose uptake.
Thanks a lot for your quick answers!
Mark Ian Fowler do you now a gentle method for permeabilization which also allows analysis of TFs in the nucleus?
Ion Bogdan Mănescu to be honest, that was also what I feared. And then there would be no solution...
Alexandr Chernov unfortunatly the link is not working for me. Is it correct?
Thanks again,
Katharina
How about using Streptolysin O to reversibly permeabilise the cells before NBDG incubation so that the antibody would already be inside the cell therefore no longer needing permanent permeabilisation? I've never tried it myself, and antibodies are at the upper limits for the Streptolysin pore but it would be a cool solution! Article Delivery of proteins into living cells by reversible membran...
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