Comet Assay as an Indirect Measure of Systemic Oxidative Stress

The overall goal of this procedure is to use the COMET assay for indirect measurement of oxidative stress. This is accomplished by first isolating leukocytes from human blood samples. The second step is to mix the cells with low melting aros and immobilize the cells on slides.

Next, the cells are lysed for two hours, followed by electrophoresis after staining the slides with a dium bromide. The DNA is visualized by fluorescence microscopy. Ultimately, DNA breaks are quantified by software analysis of the fluorescence microscopy data.

Today we know that oxidative stress plays a crucial role in the pathogenesis of variety of diseases, including of course, eye diseases. There are many different ways of measuring oxidative stress. One way to measure it indirectly is by means of comit assay.

The main advantage of this technique over existing methods is that it is a simple, low cost and reliable tactic with which we can indirectly measure oxidative stress. The Lucas leukocytes for this experiment will be isolated from human blood samples obtained with an anticoagulant by venous puncture to separate the lymphocytes, first dilute the blood in a one-to-one ratio with PBS. Then layer over with 600 microliters of a density gradient cell separator liquid, such as histo pack centrifuge at 1, 800 G for 20 minutes at four degrees Celsius.

Aspirate the Buffy coat into three to five milliliters of PBS centrifuge at 300 G for 10 minutes. To palate the lymphocytes using a pipetter, retrieve the lymphocytes from just above the boundary between the PBS and cell separator liquid. Remove the leukocyte bands from the interface between the plasmid and the cell separator liquid layers of each tube and collect into one 50 milliliter tube.

Bring the total volume to 50 milliliters with cold PBS. Wash the cell suspension three times with PBS at 300 G for 10 minutes. After the third wash count the total number of cells using a hemo cytometer.

Suspend the cells in PBS and Eloqua into micro centrifuge tubes at 10 million cells per tube pipette. Point four milliliters of cells into a 1.5 milliliter disposable plastic tube. Add 50 microliters of cell suspension into 500 microliters of a 1%low gelling temperature.

Aros at 40 degrees Celsius, prepared in advance mix and rapidly pipette 50 microliters of the cell suspension onto the agros covered surface of a pre-coated slide. Allow the agros to gel for about two minutes at four degrees Celsius. Be consistent with the time and temperature used for jelling, and ensure that the agros is fully set before proceeding to the lysis procedure.

Once the agros is fully set, submerge the slides in a lysing solution at four degrees Celsius for at least two hours. Next, gently remove the slides from the lysing solution. Place the slides side by side on a horizontal gel box near one end, sliding them as close together as possible.

Fill the buffer reservoirs with freshly made electrophoresis, buffer of pH greater than 13. Note that the procedure demonstrated here is for electrophoresis. Under alkaline conditions with pH greater than 13, fill the buffer reservoirs until the liquid level completely covers the slides.

Avoid bubbles over the aros. Let the slide sit in the alkaline buffer for 20 minutes to allow for unwinding of the DNA and the expression of alkali lab damage. After 20 minutes, turn on the power supply to 25 volts and adjust a current to 300 milli ampers by raising or lowering the buffer level.

Electrophoresis slides for 30 minutes. After 30 minutes, turn off the power. Gently lift the slides from the buffer and place them on a drain tray in a dropwise fashion coat the slides with a neutralization buffer.

Allow the slides to sit for at least five minutes. Drain the slides and repeat the coating with neutralization buffer two more times. Next, stain the slides with one milliliter of one x Ethereum bromide for five minutes.

Wear protective gloves because ahy bromide is a known carcinogen. After five minutes, dip the slides in, chilled distilled water to remove any excess stain. Then place a cover slip over each slide.

Store the slides immediately. Stain slides can be stored for a month at four degrees Celsius in dry conditions. To visualize assess DNA damage, observe the atherium bromide stained DNA slide under a 40 x objective fluorescent microscope.

Set the automated tool that scans over the microscope, slide and photograph the cells in the comet assay. DNA damage was assessed quantitatively in cells by measuring the length of DNA migration and the percentage of migrated DNA. The black arrowhead indicates a typical comet with a bright head and tail.

The head is composed of intact undamaged, DNA, and the tail is composed of smaller damaged DNA fragments. Although the COMET assay renders reproducible results, it can be influenced by a variety of factors such as whether the assay is performed on freshly prepared leukocytes. In this graph, tail moment is defined as the product of the tail length and the tail.

DNA percentage of the total DNA Group two leukocytes that were cryopreserved prior to lysis and electrophoresis show significantly higher single stranded DNA breaks than freshly prepared group one leukocytes. This assay can also be used to indirectly assess systemic oxidative stress. Here, olive moment represents the product of the percentage of total DNA in the tail and the distance between the centers of the mass of head and tail regions.

Results from age and sex matched smokers and healthy subjects reveal that smoking half a pack of cigarettes daily more than doubles the number of single stranded DNA breaks in the circulating leukocytes Once mastered. This technique has several advantages, including its relative low cost, the small number of cells, and therefore the small blood sample required per patient, as well as the relatively short period of time required to quantify DNA damage in cells.