When we used 2-NBDG for Glucose uptake in 3T3 cells, 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?
Cellular autofluorescence should not be too severe at the excitation and emission wavelengths of NBD (465, 540 nm). The NBD group is pretty hydrophobic, so it would not be surprising if it stuck to the cell membranes. I don't know how you can prevent it or wash it off. Maybe you can treat the cells briefly with a mild detergent or a low concentration of DMSO before making the measurement.
Thanks Eman.
We are using the 2NBDG at 200uM. Reasonable concentration, no?
Why would the phenol-red effect the fluorescence? The assay itself is done in PBS...So no phenol red there....Only in the culture medium....
We are still not making measurements because due to the high background, all the readings give the same results in the Fluorometer....
Only when we look through the microscope we can detect differential uptake with our test item.
Hi Adam. Thanks!! our 2NBDG was dissolved in DMSO!! Could this be (counter-intuitively) the reason for the unspecific binding to the Glucose to the cells?
DMSO is a solvent that can affect the permeability of lipid membranes. If the concentration was high enough, it might have allowed 2NBDG to get into the cells.
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