Hi here's a general protocol I have used for radiolabelled 2-DG. The principle will be very similar. I typically used 10uM 2-DG for my uptake buffer. The key will be optimising the relative concentration of labelled and non labelled 2-DG that gives a good signal in the linear range.

seed cells in 24 well format at 5x10^4 -wait 24 hours

Serum starve cells in PBS +5mM glucose for 3 hours prior to assay start

Add insulin or DMSO control at concentration of 100nM for 30mins

Beigin assay as below-

1. Aspirate media from cells.

2. Wash cells 2x HBS.

3. Aspirate HBS from fist plate and add uptake buffer (1ml in 12 well plate).

4. Leave for 10 minutes

(start next plates after every 2 mins)

5. Aspirate uptake buffer and wash 2x 0.9% NaCL, do this as quickly as possible.

6. collect cells, spin down and re suspend in an appropriate ice cold buffer (low serum buffer suitable for flow cytometry).

Analyse by flow cytometry.

1X HBS buffer

4.8g (2.4g) HEPES pH 7.4 (20mM)

8.2g (4.1g) NaCl (140mM)

1.2g (0.6g) KCL (16mM)

616mg (308mg) MgSO4 (5mM)

147mg (74mg) CaCl2 (1.3mM)

H2O to 1L (500ml)

You can see our radiolabelled work in skeletal muscle here -

Article Multiple AMPK activators inhibit L-Carnitine uptake in C 2 C...

Alternatively check below for a basic protocol from a kitfor your assay that you can buy. It provides all the buffers you would need.

https://www.biovision.com/documentation/datasheets/K682.pdf

Finally here is a paper that has done exactly what you are looking for.

Article 2-NBDG as a fluorescent indicator for direct glucose uptake ...

Hi Andrew

i detailled below the protocol i used to induce insulino-resistance and assay 2-NBDG cell incorporation. Unfortunally, the values that i obtained was the same. is there any modifications i should do?

2-NBDG glucose uptake assay on insulin-resistant HepG2 cells

- Culture cells (2 x 104/well) in a black clear bottom 96-well microtiter plate in a final volume of 100 µL/well in presence of additional factors for viability/proliferation testing.

- Incubate cells overnight or 80-90% of confluence. The appropriate incubation time will depend on the individual cell type and cell concentrations used.Therefore, it is recommended to determine the optimal incubation time for a particular experiment.

- Carefully remove (decant) the overnight culture medium from adherent cells and add in each wells 100µL of fresh medium (without growth factors: FBS) containing 1 µM insulin in serum-free MEM (5.5 mM Glucose)

- Incubate the plate at 37°C for 24 hours in a humidified, 5% CO2 atmosphere

- Carefully remove (decant) the overnight culture medium from adherent cells and add in each wells 100µL of fresh medium low glucose (without growth factors: FBS) containing for each well the amount of sample (concentration) need for the experiment

- Incubate the plate at 37°C for 24h in a humidified 5% CO2 atmosphere

- Carefully remove (decant) the culture medium containing samples from adherent cells and add in each wells 100µL of fresh medium low glucose (without growth factors: FBS) containing 0.1 µM insulin in serum-free MEM (5.5 mM glucose)

- Incubate the plate at 37°C for 30 minutes in a humidified 5% CO2 atmosphere

- Then, add in each wells 100 µL of 2-NBDG (40 µM) prepared on PBS (1% BSA)

- Incubate the plate at 37°C for 30 minutes in a humidified 5% CO2 atmosphere

- Washed cells two times with 200 µL of ice-cold PBS to stop uptake

- Add in each wells 100 µL of PBS

- Measure the 2-NBDG fluorescence intensity on a fluorescence microplate reader at 465 nm excitation and 540 nm emission wavelengths.