2-NBDG

1.1 Preparation of the stock solution
Dissolve 1 mg of 2-NBDG in 2.92 mL of DDH2O to obtain 1 mM of 2-NBDG.
Note: It is recommended to store the stock solution at -20 ℃ or -80 ℃ away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of 2-NBDG working solution.
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 10-200 μM of 2-NBDG working solution.
Note: Please adjust the concentration of 2-NBDG working solution according to the actual situation.
Cell staining
2.1 For suspension cells: Centrifuge at 1,000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4℃ for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of 2-NBDG working solution, and then incubate at room temperature for 5-60 minutes.
2.3 Centrifuge at 400 g at 4℃ for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS. .If test viability, recorded the optical density (O.D.) at 540/570 nm. Cell viability was calculated as a control ratio and plotted against the logarithmic concentration of the drug to calculate IC50.

Nine culture plates of astrocytes are used for each uptake experiment. After medium is removed and plates are rinsed in wash buffer, cells are incubated at 37 °C in buffer containing 25–200 μM 2-NBDG. After 1 h, buffers are replaced by fresh incubation buffer with 2 mM glucose and incubation is continued for 5 min. The cells are washed twice with prechilled phosphate-buffered saline and are incubated at room temperature in 0.1 mL cell lysis buffer for 10 min.

2-NBDG fluorescence intensity following 30-minutes topical application was 6-fold and 4-fold higher in OSCC and OED, respectively, compared to normal mucosa. Receiver operator characteristic analysis show 83% sensitivity and 73% specificity for detection of neoplasia vs benign (normal and inflammation). Faster 2-NBDG fluorescence temporal decay in neoplasia indicated higher uptake and glucose metabolic rate than normal mucosa. Mucosal delivery of 2-NBDG by topical application to the in-vivo oral surface is feasible and delineates neoplasia from normal mucosa, providing in-vivo noninvasive molecular imaging of dysregulated glucose metabolism.^[2]