2-NBDG is a fluorescent indicator for direct glucose uptake measurement. It is an indicator of cell viability.

METHODS: Flow cytometry was used to detect glucose uptake:
1, Cells were seeded at 1*104/well in 96-well plates, and the experiment was performed within 24-48 h. The cells were incubated at 37 ℃ for 10-180 min.
2, Remove the cell culture medium, add fresh medium containing 2-NBDG (5-40 µM), and incubate for 10-180 min at 37 ℃.
3. Remove the medium and wash twice with pre-cooled PBS to stop the 2-NBDG uptake reaction. Resuspend in fresh medium and perform flow cytometry within 30 min. [1]

METHODS: Glucose uptake by circulating breast cancer cells was detected by fluorescent microscopy:
1. A mouse blood sample (100 µL/mouse) was collected by puncturing the mouse saphenous vein.
2. Incubate the blood sample containing circulating breast cancer cells with 2-NBDG (5 µg/100 µL blood) for 30 min at 37℃ in a dark incubator.
3. Add the magnetic bead suspension (1µL 1%) to 100 µL of blood sample and incubate for 30 min at 4°C with gentle shaking to promote binding of the magnetic beads to the circulating breast cancer cells.
4. Separate the circulating breast cancer cells from the blood using a magnetic separation rack, wash with PBS 3 times, resuspend in 100 µL PBS and transfer to a 96-well cell plate.
5. 2-NBDG uptake by circulating breast cancer cells was examined under a fluorescence microscope equipped with a 488 nm filter. Large cells with a fluorescent signal derived from fluorescent 2-NBDG uptake by cells were counted as hypermetabolic circulating breast cancer cells, and small-sized normal mouse blood cells (lymphocytes and RBCs) showed no or little 2-NBDG fluorescent signal. [2]

Flow cytometry to detect glucose uptake
Operation steps:
a. Cells were seeded in 96-well plates at 1*104/well and the experiment was performed within 24-48 h;
b. Cell culture medium was removed, fresh culture medium containing 2-NBDG (5-40 µM) was added, and incubated at 37 ℃ for 10-180 min;
c. Culture medium was removed, and the cells were washed twice with pre-cooled PBS to stop the 2-NBDG uptake reaction. Resuspended in fresh culture medium, flow cytometry was performed within 30 min.