Experimental example: Enhanced glucose uptake by insulin
In diabetes research, the glucose uptake can provide important information for understanding glucose metabolism and regulation. Screening of drugs that regulate glucose uptake and evaluation of insulin resistance are important to treat diabetes.
[Protocol]
1. Adipocyte in DMEM (10% FBS) were seeded in a 96-well microplate.
2. After removing the supernatant, the cells were washed twice with DMEM (5.5 mmol/l Glucose, Serum-free).
3. After removing the supernatant, DMEM (5.5 mmol/l Glucose, Serum-free) containing 1,000nmol/l Insulin was added, and the cells were incubated at 37°C 15 minutes.
4. After removing the supernatant, Probe solution in DMEM (Glucose-free, Serum-free) was added and the cells were incubated at 37°C for 15 minutes.
5. After removing the supernatant, the cells were washed three times with WI Solution at 4°C.
6. The cells were observed under a fluorescence microscope and the fluorescence intensity was measured with a microplate reader.
Insulin concentration
Insulin incubation time
Please perform insulin treatment using 1,000 nM Insulin for 15 minutes during your first attempts.
Longer insulin treatments (24 hours) show no difference in glucose uptake.
[Technical Tips]
・Especially with adipocyte samples, we recommend first testing the probe using un-stressed cells.
・Fasting/starvation can be performed in later experiments.
・Please perform insulin treatment prior to – and separate from – probe incubation.
・Please perform insulin treatment using 1,000 nM Insulin for 15 minutes during your first attempts. Longer insulin treatments (24 hours) show no difference in glucose uptake.
・First time users are recommended to perform probe incubation for 15 minutes and only introduce glucose-free medium at that stage.
・First time users are recommended to perform 500-fold probe dilution, as suggested by the manual.
・Please wash with WI solution at least 3 times and measure shortly after the procedure.
