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PFA

find the below attachment

Procedure 1. One day previous to infection, prepare 48-well dishes by seeding each well with 7 x 104 cells in 0.5 ml DMEM plus 7.5% fetal bovine serum, 4 mM glutamine, antibiotics. Alternatively another cell density may be required, based on the cell line required for viral growth. 2. On the day of infection, make dilutions of virus sample in PBS. For influenza virus, use cell media plus TPCK trypsin for dilutions. - Tips used for virus transfers: Aerosol resistant 20 μl, 160ul and 1000 μl sterile tips. 3. Make a series of dilutions at 1:10 of the original virus sample. Fill first tube in series with 2.0 ml of PBS, fill the remaining 6 tubes in series with 1.8 ml of PBS. 4. Vortex virus sample, transfer 20 μl of virus to first tube, vortex, discard tip. 5. With a new tip, transfer 200 μl of first dilution to next tube. Vortex, discard tip. 6. Repeat series of dilutions through the last tube. Additions of virus dilutions to cells: 1. Label lid of 48-well dish by drawing grid lines to delineate quadruplicates and number each grid to correspond to the virus sample and label the rows of the plate for the dilution which will be plated. 2. Include four negative wells on each plate which will not be infected. 3. Remove all but 0.1 ml of media from each well by vacuum aspiration. For influenza virus, monolayers should be rinsed once with serum free media before 0.1 ml infection media is added per well. 4. Starting from the most dilute sample, add 0.1 ml of virus dilution to each of the quadruplicate wells for that dilution. 5. Be careful not to track the pipette tip over the wells, and continue infecting the plate with 0.1 ml of virus dilution per well, infecting four wells per dilution, proceeding backwards through the dilutions. 6. Change the pipette tip when necessary. 7. Allow virus to adsorb to cells at 37o C for 2 hours or at a temperature indicated for the specific virus being titered (some viruses grow better at 34o C, including influenza). 8. After adsorption, remove virus inoculum with vacuum, beginning by sucking the most dilute wells and proceeding backwards to less dilute. Alternatively, leave the virus on the cells. 9. Add 0.5 ml Infection Medium (DMEM, 2% FBS, 4 mM Glutamine, 1X PSF) to each well. Do not touch the wells with the pipette. For influenza virus, add Infection Media without serum, with TPCK trypsin. 10. Place plates at 37o C or 34o C, depending upon instructions and monitor CPE using the inverted microscope over a period of one to four weeks, depending on the cultural characteristics of the virus in question. 11. Record the number of positive and negative wells. 12. Calculate the TCID50 titer using the Excel spreadsheet using below attachment