How long are you starving your cells? Are you washing your cells to remove free 2NBDG? 

• Keep your cells for 12 hours in serum starvation using DMEM + 0.1% BSA
• wash cells with Krebs Ringer Phosphate Buffer supplemented with 0.2% BSA
• incubate the cells for 1 hour at 37oC
• stimulate with insulin (200 nM) and 2NBDG (80uM) for 30-60 min
• wash cells with Krebs Ringer Phosphate Buffer supplemented with 0.2% BSA

Dear Andre.F,Thank you for your reply.

I have starved my cells for more than 12 hr with DMEM+0.1% Fetal Bovine Serum. I used to wash my cells with filtered PBS (PH-7.4)for almost 3-4 times before going for FACs,I have standardized the concentration of Dye (2-NBDG) from 5uM to 50 uM and found good effect at 10 uM .Trying all the possible methods mentioned above,still I find no effect in the glucose uptake with insulin.

Hi Soheb

Both cells give the same result? Neither one has at least a small increase? Can you walk me through your flow cytometry steps? 

• doublet exclusion
• viability
• gating

Are you staining your cell for something other than glucose uptake?

Hello,

I have tried in all the possible ways with H9C2 (cardiomyocytes) but just twice I have checked with HepG2 cell line, unfortunately I did not notice any marked differences between these 2 cell lines.

The Protocol for FACs which I follow is:

1.I grow the cell in High glucose DMEM (25mM) with 0.1% fetal bovine serum for 12 hrs before checking the 2NBDG uptake.

2.Remove the media wash with PBS for 2-3 times. 

3.Incubate 2NBDG in PBS for one hour.

4.Wash the cells with Ice cold PBS 3-4 times.

5.Tripsinized  and neutralize the cells and take for Facs.

6.I have never tried to check the live dead cell population with any dye.

Thank 

Hi Soheb

I believe the problem is that you are not glucose starving your cells before adding 2NBDG. You should use glucose free media 1-16h (depending on the cell) before adding  2NBDG. Timing of incubation should be long enough to show sufficient differences in the end result.

Hii Andre.F

Yes am not starving my cells with glucose, so in my next experiment I will use glucose free media to check glucose uptake.

Kind regards

soheb 

Hii,

I did not tried yet,once am done I will get back to you with the results.

Kind regards

sohebi

i have met the same problem like you.

So after all did you won the experiment? tring to replicate but i felt

Has anyone performed the glucose uptake in A549 cell line.