The overall goal of this procedure is to titrate glycogen in vitro in cell samples. This is accomplished by first lysing the cells by mechanical disruption. The second step is to hydrolyze the glycogen with alpha amylase to obtain the glucose.
Next, the glucose is oxidized producing a fluorescent species. The final step is to measure the fluorescence. Ultimately, biochemical titration of glycogen is used to show changes in glycogen content in cells.
The main advantage of this technique over existed methods is that it is competitive, sensitive, accurate, and very specific. To begin this procedure, seed cells at a concentration of 0.5 to 2 million per 100 millimeter diameter dish. Incubate the cells for 24 48 or 72 hours in hypoxia in an anaerobic workstation set at 1%oxygen, 94%nitrogen and 5%carbon dioxide.
In parallel, incubate another set of cells in normoxia for each oxygen condition. Change the 25 millimolar glucose containing medium every 24 hours to minimize variations in the glucose concentration during the experiment after treatment. Wash the cells twice with PBS to remove traces of glucose from the culture medium.
Then scrape the cells in cold PBS centrifuge, the PBS solution at 1000 RPM for five minutes. Discard the supernatant and wash the pellet once with PBS. Next, re suspend the pellet in 100 microliters of distilled water and add 100 microliters of the glycogen hydrolysis buffer obtained from a glycogen assay kit.
Boil the lysate at 95 degrees Celsius for five minutes to inactivate the enzymes. Once the lysate is cooled to room temperature, vortex and centrifuge. The lysate at 13, 000 times.
Gravity for 10 minutes to remove the insoluble products. Then set aside the supernatant to normalize glycogen levels to the protein content between samples. Quantify the proteins with 25 to 35 microliters of the supernatant using the bsic acid assay.
Following this mix, 50 microliters of the supernatant from the previous steps with one microliter of the hydrolysis enzyme mix. Incubate the mixture for 30 minutes at room temperature and set aside to prepare the calibration curve standard, dilute the standard kit glycogen with hydrolysis buffer to give a final concentration of 10 micrograms per milliliter. Next, add 0, 4, 8, 12, 16, and 20 microliters of the 10 microgram per milliliter.
Standard to six separate tubes dilute each standard with hydrolysis buffer to give a final volume of 50 microliters and a glycogen concentration of 0.04, 0.08, 0.12, 0.16, and 0.2 micrograms per milliliter respectively. Add one microliter of hydrolysis enzyme mix to the standards and incubate for 30 minutes at room temperature for each oxygen condition. Label tube one as free glucose concentration and tubes two and three as total glucose concentration.
Then add 15 microliters of the extracted supernatant to tube one followed by 35 microliters of the hydrolysis buffer to give a final volume of 50 microliters. The fluorescence corresponding to the free glucose concentration will be measured later. Add four microliters of the hydrolyzed glycogen to tube two and adjust to a final volume of 50 microliters with hydrolysis buffer.
To keep the fluorescence values within the concentrations of the calibration curve, add two microliters of hydrolyzed glycogen to tube three and adjust to a final volume of 50 microliters with hydrolysis buffer. Next, prepare a development solution for all samples and standards by mixing 48.7 microliters of development buffer, one microliter of development enzyme mix, and 0.3 microliters of Oxy Redd Probe. For each sample standard, add 50 microliters of this mix to each tube and incubate in the dark for 30 minutes.
At room temperature for the fluorescence measurement, set the slit to three nanometers for excitation and emission, and then measure the fluorescence. The regulation of glycogen storage and hypoxia was studied as glycogen is the main energetic polymer of glucose in mammalian cells. The calculation and standardization of the glycogen concentration in cell sates was performed on the raw data of fluorescence.
As shown here from the fluorescent measurements, the total free glucose concentration was calculated. Using these results, the glucose amount derived from glycogen was determined. The biochemical assay for glycogen confirmed that the electron dense aggregates observed on electron micrographs of ccl 39 cells were glycogen particles.
The accumulation of glycogen and hypoxia is dependent on the transcription factor. Hypoxia inducible factor one, the major transcription factor involved in cellular adaptation to hypoxia in different cancer and non-cancer cell lines stored glycogen can be rapidly metabolized by the cell in less than six hours. In addition, the use of glycogen protects against cell death under conditions of glucose starvation Following this procedure.
Also, methods like electron microscopy and periodic acid shift staining can be performed in order to answer additional questions like the distribution of glycogen in cells.
