Hello Ayman Banah

Try using the protocol provided in the research article attached. You could modify the protocol according to your need.

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2-NBDG Uptake of H9C2

H9C2 was incubated with 2-NBDG, which is a fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose, to investigate the glucose uptake ability.

H9C2 was seeded in six-well plates. After hypoxia-reoxygenation— (a Hypoxia-reoxygenation cell model was used in our study. Briefly, after serum-starvation for 18 h and then treating with different concentration of agents, H9C2 was subjected to hypoxia in a controlled hypoxic plastic chamber for 12 h. Reoxygenation was conducted in a normoxic incubator at 37°C after hypoxia for 4 h), cell culture was replaced by Kreb's buffer without glucose (NaCl [145 mM], KCl [5 mM], CaCl2 [6 mM], MgCl2 [1 mM], HEPES Na [25 mM], and NaHCO3 [10 mM], pH 7.4) and incubated with different concentrations of LPA and insulin for 20 min at 37°C.

Thereafter, 2-NBDG (100 μM) (Invitrogen, California, USA) was supplemented at the same culture temperature for an additional 10 min in the dark. Fluorescence was measured in a FACS Calibur Analyzer (BD) by 488 nm (excitation wavelength) channel. Data was collected from 20,000 events for each measured sample.

Hope this is helpful.

Good Luck.

Kindly refer the protocol and adjust it according to your findings, it might become a reference for your experiment.

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