The overall goal of this procedure is to demonstrate how to use the COMET chip to perform high throughput DNA damage measurements in mammalian cells. This is accomplished by first loading mammalian cells into the COMET chip. The second step is to treat cells with chemicals that are known to cause DNA damage.
Next, the cells are lysed and Electro East using traditional COMET assay protocols. The final step is fluorescence imaging of cellular DNA to visualize the migration of damaged DNA. Ultimately image analysis software is used to quantify the extent of DNA damage.
So the com assay has been around for a really long time and it's a very useful assay for looking at lots of different kinds of DNA damage. But the problem has been that it's really low throughput and it's lacks sensitivity. So we created the COMET chip, which basically is a very high throughput version of the COMET assay that makes it possible to do high throughput screening for drug screening, for example, and for epidemiological studies.
Jing GA graduate student in my laboratory will be demonstrating the procedure. The coma chip is a gel with an array of micro wells produced from a micro fabricated stamp with micro posts with diameters that are as small as that of a single cell to begin place into a one well rectangular plate gel bond film side down. After washing the gel twice in 25 milliliters in one XPBS set the comma chip on a glass plate, then label the orientation of the gel accordingly.
Now gently press an inverted bottomless 96 well plate into the gel ensuring that all 96 wells are within the area of the gel. Then clip either side of the 96 well plate to the glass plate with 1.5 inch binder clips. Lastly, aspirate off any excess PBS from each.
Well harvest suspension or adherent cells according to standard procedures and dilute the cells with media to a final concentration of 100, 000 to 1 million cells per milliliter. Ensure that a single cell suspension is obtained by passing through a cell filter if necessary. Pipette 100 microliters of cell suspension into each well, the comet chip 96 well plate when finished.
Cover the plate with the gel bond film to prevent evaporation, and then place in an incubator set at 37 degrees Celsius for 30 minutes after removing the plate from the incubator. When the 30 minutes have elapsed, aspirate the media from each, well then remove the binder clips and the bottomless 96 Well plate Hold the comet chip with the glass plate at an angle and gently rinse with one XPBS to remove any excess cells on top of the aros. Now view the plate through a four x objective lens on a bright field microscope to check for optimal cell loading After sufficient cells are loaded, placed a com chip on an even surface, an overlay with 1%low melting point agros that has been prewarm to 37 degrees Celsius.
Allow the overlay to solidify at room temperature for three minutes and then place it four degrees Celsius for five minutes for complete dation to dose the cells with chemicals of interest. First, carefully align the wells of the comet chip with the wells of a new bottomless 96 well plate and press down. Secure the 96 well plate to the glass plate with binder clips as before.
Then place the plate on ice and add 100 microliters of the chemical of interest at the desired dose To each well allow the chemical under investigation to remain on the cells with the desired period of time after dosing, aspirate the chemical solution from each well and rinse with one XPBS if required. Cut the gel into separate pieces. Then to study repair kinetics, add culture media to the wells incubate and proceed to cell lysis at specific time points to LY cells for the alkaline comet assay.
Prepare approximately 25 milliliters of working alkaline lysis buffer for each chip by adding 1%Triton X 100 to the alkaline lysis stock solution. Then pre chill at four degrees Celsius. Decant the alkaline lysis buffer into a container just larger than the comet chip.
Then submerge the alkaline chip in the chilled working alkaline lysis buffer place in the refrigerator and allow lysis to proceed overnight at four degrees Celsius. The next day, remove the comet chip from the lysis buffer and quickly rens with one XPBS. Then place the chip in an electrophoresis chamber with the gel film side facing down and secure with double-sided tape.
Take the chamber to the four degrees Celsius cold room and fill the chamber with cold alkaline electrophoresis buffer to a level that just covers the gel. Then leave the chip at four degrees Celsius for 40 minutes to allow for alkaline unwinding. Finally, run the gel at one volts per centimeter and 300 milliamps for 30 minutes.
In the cold room, adjust the volume of the electrophoresis buffer in the chamber to achieve desired running current. For the neutral comet assay, make working neutral lysis buffer by adding 1%Triton X 110%DMSO to neutral lysis stock solution. Again, preparing approximately 25 milliliters of working lysis buffer for each chip.
Place the working neutral lysis buffer in the incubator set to 43 degrees Celsius to prewarm. After the neutral lysis buffer is warmed, submerge the comma chip in the neutral lysis buffer and place in a 40 degrees Celsius incubator overnight. The next day, remove the neutral lysis buffer and then res twice with neutral electrophoresis buffer for 15 minutes at four degrees Celsius Each time, secure the chip in the electrophoresis chamber and transfers to the cold room.
As before, fill the electrophoresis chamber with cold neutral electrophoresis buffer to a level that just covers the gel and allow the gel to sit in a cold room for 60 minutes, run the gel at 0.6 volts per centimeter and six milliamps for 60 minutes in the cold room. Again, adjust the volume of the electrophoresis buffer in the chamber to achieve desired running current if needed. After removing the com chip from the cold room, neutralize the gels by washing and neutralization buffer twice for 15 minutes each at four degrees Celsius.
Stay in the chip with a fluorescent DNA stain such as cyberg gold or iridium bromide. According to the manufacturer's instructions and image using fluorescent microscopy representative Microwell comets from untreated TK six lymphoblasts are shown in the top panel of this diagram. Those from TK six cells exposed to 50 micromolar hydrogen peroxide in the middle panel and 100 micromolar hydrogen peroxide in the bottom panel.
All exposures were for 20 minutes at four degrees Celsius. This line graph shows the hydrogen peroxide dose response of TK six cells. Each data point is the average of three independent experiments where the median percentage tail DNA of at least 50 individual comets were obtained in each experiment.
This graph shows sample to sample variation of the comma chip assay for TK six cells exposed hydrogen peroxide percent tail DNA data of each well is shown here. Each box represents the median at least 50 individual comets from each well. The mean of 12 wells is 77 point 53%Standard deviation is 3.99%and the coefficient of variation is 5.2%This graph shows experiment to experiment variation.
The same hydrogen peroxide exposure was repeated six times, and data of each repeat is shown as a gray bar. Each box represents the median percent tail DNA of at least 100 individual comets from each repeat. The average of six repeats is 49.57%shown as the back bar here.
The standard deviation of six repeats is 4.99%and the error bar of the mean is 2.04%represented as the air bar in the figure Like the traditional common assay. Researchers are free to make modifications to this procedure or use other messes, such as including purified lesion specific enzymes to answer additional questions about the type of DNA damage produced in cells. The comet chip technique has paved the way for studies of mammalian cells to learn about DNA damage and repair.
And because it's high throughput, it makes it possible to do clinical and epidemiological studies that just weren't possible before due to the number of samples.
