It is possible to avoid use of radioactive non-metabolizable glucose analogs by using a fluorescent tagged D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG). Detection and quantitation can be performed by flow.
A large fluorophor hanging off the side may have some effect on the kinetics but that's the price!
http://www.sciencedirect.com/science/article/pii/S0165022X05001260
What about measuring the glucose which is left in the medium? If you only have cells present in the medium, they are the only ones consuming glucose.
Tausif Alam is correct, you can also measure uptake with 2nbdg with confocal, the protocol is pretty identical to 3h-2dg uptake. alternatively you can measure glut4 trans-location to the cell surface, as some drugs can increase translocation but actually bind to glut4 itself to inhibit glucose uptake (such as forskolin).
Dear Dana,
Thanks for your reply. Actually, I am interested to study the Glut1 mediated glucose uptake (i.e. basal level uptake). Also, can you suggest how to use flow cytometry for the same.
i havent done flow for 2nbdg but many others (esp in cancer field) have, its a florescent ligand so you can easily sort just on flourescence basis/scatter as you dont need an antibodies
you can also use it for glut1 uptake, would suggest to do sirna for glut1 to make sure its that then
I will be using MDA-MB-468, HEK293t and HeLa cells.
Also, there is a Cayman kit available which uses 2-NBDG. Will it help?
we have done 2nbdg in a few cell types, the closest would be cho cells for you, it worked perfect and we got the same answers as 3h-2dg (unpublished data). i would expect a big difference in basal gluose uptake in those three cell lines, esp the mda cells
from a quick look at the kit, im a bit concerned of their protocol thats says to do the epxt in glucose free media for 10mins-overnight with 2nbdg. 2nbdg is still an analogue of 2-dg, so once that gets inside the cell, it cant do anything, cant go into glcolysis etc so the cells may start to die, so a short time period may be more realistic (for confocal we did 10min). we bought 2nbdg from life tech and just substituted it for the 3h-2dg in our preexisiting conditions (albeit on slides for confocal rather than plates)
you should try it, the main concerns are the time and concentration you need. the original papers from japan had a lot of information about that included but they did (from memory) pancreatic cells in their papers
you will have optimise those 2 main conditions but for not using radioactivity (and that method can have many limitations) its worthwhile to establish this method
Hey, I got it. so, you think its better to buy 2nbdg from life tech and use in the experiments. Can you send me the links for the original papers from Japan.
When we used 2-NBDG, we got alot of background fluorescence (Perhaps due to binding to the membrane outside??) This made quantifying uptake using a fluoremeter impossible, albeit obvious differences when observed in the microscope.
-Is there non-specific binding of 2-NBDG?
Is there auto-fluorescence of 3T3 cells?
Is there a method to efficiently wash away the non specific binding of 2-NBDG?
we have tried with plate readers and get no logical results, but with confocal it works perfect, then you just have to figure out a way to quantify it
hence we dont do plate readers/fluorometer, but other people have done FACS sorting and it works and then you can quantify it
2nbdg can be easily used for glucose uptake assays in vitro by using a fluorimeter capable of bottom probe reading. You will need 96 well black plates with clear bottom. I use this assay routinely and have used it in couple of publications recently.
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