This method will allow us to isolate Epstein Barr virus particles from the P3HR1 cell line and quantify the virus stock so that it can be used for various research purposes. The main advantage of this technique is that it provides a relatively easy, inexpensive, fast, and reliable method to prepare an EB viral stock. The vital particles produced by this technique can be used in many in vitro or in vivo experimental models for the diagnosis of EB.To begin, prepare a volume of cell suspension A in a 15 milliliter conical tube, adjust the volume such that the number of cells is roughly 2.2 million cells for a 100 millimeter culture plate.
Add five milliliters of complete culture medium to the tube. Add 80 microliters of dimethyl sulfoxide to the tube. Then add 350 microliters of one milligram per milliliter PMA to the tube.
The final concentration of PMA should be 35 nanograms per milliliter, and that of DMSO should be 0.08%Add 4.27 milliliters of culture medium so that the total volume is 10 milliliters. Mix the contents of the tube by tilting, then transfer the contents to a 100 millimeter culture plate. Leave the plate in a cell culture incubator for five days at 37 degrees Celsius, 5%carbon dioxide.
After five days in the incubator centrifuge the contents of the plate at 120 G for eight minutes to pellet the cells Collect the cell-free virus containing supernatant and discard the cell pellet. Again centrifuge the supernatant at 16, 000 G for 90 minutes at four degree Celsius to pellet the virus particles. Discard the supernatant and resuspend the virus pellet in five milliliters of culture medium, Aliquot the viral suspension into 20 tubes each containing 250 microliters of the viral suspension, and store the suspension at minus 80 degrees Celsius.
To separate the proteins from DNA add 500 microliters of Tris-saturated phenol to one of the tubes containing the viral preparation. Add 100 microliters of water and vortex well to obtain a pink emulsion. Centrifuge for 15 minutes at 9, 650 G, collect the supernatant and transfer it to a new 1.5 milliliter micro centrifuge tube.
Add an equivalent of one-tenth of the supernatant volume of cold sodium acetate and mix by pipetting up and down. Then add one microliter of 20 milligrams per milliliter glycogen, and mix by pipetting. Add three times the supernatant's volume of cold 100%ethanol and store it at minus 80 degrees Celsius overnight.
To isolate the viral DNA centrifuge at 9, 650 G for 15 minutes at four degrees Celsius. Discard the supernatant to obtain a DNA pellet and wash the pellet three times with one milliliter of cold 70%ethanol. Again centrifuge at 9, 650 G for 15 minutes and discard the supernatant.
After air drying the pellet for about 10 minutes, resuspend the pellet in 10 to 50 microliters of nuclease free distilled water. Store the samples at minus 20 degrees Celsius for later processing or at four degrees Celsius overnight to ensure maximal DNA dissolution, followed by storage at minus 20 degrees Celsius. After cleaning the pedestal of a micro spectrophotometer with a delicate task wiper load one microliter of the prepared DNA sample and note the concentration of the DNA in the sample.
Check the ratios of absorbance at both 260 to 280 nanometers, and 260 to 230 nanometers. DNA will absorb at 260 nanometers, proteins at 280 nanometers, and organic substances such as phenol will absorb at 230 nanometers. A ratio of 1.8 to 2 is considered sufficient for realtime PCR.
To prepare the PCR reaction mixes, place five microliters of a cyber green realtime PCR mix in 0.2 milliliter PCR tubes. Add one microliter of 7.5 pico moles per microliter forward primer and one microliter of 7.5 pico moles per microliter of reverse primer to each tube. Here, EBV small RNA 2 gene is amplified.
To determine the EBV genome copy number. Then add two microliters of nuclease free water and one microliter of viral DNA to one of the tubes. The total volume of the reaction mixture is 10 microliters.
Prepare other tubes that will be used as standards with known EBV genome copy numbers. Run the PCR mixtures starting with an initial step of activation at 95 degrees Celsius for five minutes. Then 40 cycles at 95 degrees Celsius for 15 seconds, and at 58 degrees Celsius for 30 seconds.
Export all data sheets as CSV files and calculate the log of the number of EBV genome copies per standard tube and mean CQ values. Generate a qPCR standard curve by plotting the CT values of the standards against the log of the number of genome copies. Employing the standard curve plot equation, derive the number of EBV genome copies in the PCR tube containing the induced viral DNA.
Finally, use the formula shown on the screen to calculate the concentration of the induced viral preparation. Here X is the number of EBV genome copies derived from the standard curve, and F is the dilution factor used to set up the DNA per PCR reaction. Cell counting using a hemocytometer chamber is shown here.
Four quadrants are counted using a light microscope. Here cells indicated in blue should be counted, while cells in red should not be counted, since they are touching the top, right, bottom, or left borders of the quadrant. An optimization trial was performed to determine the concentrations of PMA and dimethyl sulfoxide that would yield the highest number of EBV particles.
A DMSO concentration of 0.8%and a PMA concentration of 35 nanograms per microliters were optimal and resulted in high EBV concentrations. Our lab used the vital particles produced by this technique to examine pro autoimmune or inflammatory responses to this virus. As well as to develop novel anti-EBV agents.
