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TCID50 Assay Protocol
Plaque assay is an extremely useful approach for determining viral titers, however, there are several virus types which do not form plaques in culture. Alternative procedures such as TCID50 is performed to determine the infectious titer of any virus which can cause cytopathic effects (CPE) in tissue culture over a reasonable period of 5 to 20 days. In order to see a cytopathic effect, the cell line and virus must be carefully matched as not all virus types cause CPE in tissue culture.
TCID50 is the tissue culture infectious dose defined as that dilution of virus required to infect 50% of the cell monolayers. It is determined in replicate cultures of serial dilutions of the virus sample. TCID50 is widely used for plethora of application both in experimental and diagnostic virology including HIV-1, influenza and human herpes virus.

BrainVTA performs the TCID50 assay using tenfold serial dilutions of the virus sample, and plates those samples in 48-well tissue culture dishes. 
The generic procedure of TCID50 assay as follows.
1. The day previous to infection, prepare 48-well dishes by seeding each well with 7 x 104 cells. Alternatively another cell density may be required, based on the cell line required for viral growth. Gently sways plates, so that cells are distributed evenly. Next day, you should confirm that cells are evenly distributed and reached over 80% confluency under a light microscope.
2. Making tenfold serial dilutions of the virus sample. Fill first tube with 2.0 ml of PBS, fill the remaining 6 tubes in series with 1.8 ml of PBS. Transfer 20 μl of virus to the first tube and vortex gently. Then transfer 200 μl of the diluted virus to the second tubes in series.
3. Label lid of 48-well dish by drawing grid lines to define quadruplicates and number each grid to correspond to the virus sample and label the rows of the plate for the dilution that will be plated. Include four negative wells on each plate that will not be infected. Carefully remove 0.1 ml of media from each well and add 0.1 ml of virus dilution per well. Allow virus to adsorb to cells at 37°C for 2 hours (some virus types grow better at 34°C) followed by adding 0.5 ml infection medium to each well and place plates back to the CO2 incubator at 37°C or 34°C for monitoring CPE for one to four week.
4.  Calculate the TCID50 titer.

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