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8-Dilution 24-Hour Fluorescent Virus Titration Assay (96-well plate) 8-Dilutions, up to 6 virus stocks Day 1 Make 96-well plate(s) of HeLa cells to be 80-90% confluent tomorrow. One plate can assay up to 6 virus stocks. Day 2 Using 2 columns per sample, make 5-fold serial dilutions of virus in a blank 96-well plate (80l clear DMEM/7.5% FCS + 20l virus) as described below: FIRST, using an 8-channel pipettor, put 80l clear DMEM/7.5% FCS into each well of the blank 96-well plate(s). Add 20l straight virus into wells of Row A (2 wells for each sample) using a P-200 and sterile tips. Once all initial dilutions are set up, switch back to the 8-channel pipettor. Using only 6-tips, make the 5-fold serial dilutions by transferring 20l diluted virus from Columns 1-6, Row A to Row B. CHANGE TIPS! Mix by pipetting up and down 8-10 times, then transfer 20l diluted virus from Row B to Row C. CHANGE TIPS! Continue down the plate until row H is mixed. Change tips after dropping each 20l virus, then with the new tips, mix & transfer for each new dilution. When Row H is complete, Change tips again and Repeat with Columns 7-12, Row A. Remove growth medium from the HeLa cells plate: starting at Row H, remove the medium from columns 1-6, then move up through Rows G, F, & E; Add 50l from appropriate wells of dilution plate to the cell plate. Now remove medium from Row D 1-6 up to Row A 1-6; then add the virus from the dilution plate. Use the same tips to add the virus when moving up from Row H 1-6 to Row A 1-6, then change tips for Row H 7-12 through Row A 7-12. Take note of the time the plate is put back into the incubator. Incubate the infected plate overnight at 37oC. At approximately 20-22 hours for GFP Virus, or 22-28 hours for RFP Virus, check on the Inverted Fluorescent Microscope. Count the pairs of wells with over 10, but less than 100 infected (fluorescent) cells or clusters of cells (a cluster/plaque is counted as ONE infectious unit). The formula for the Virus Titer is: # Infectious Units (plaques or cells) X inverse of the dilution Divided by the volume of inoculum [if 50l inoculum, divide by 0.05 ml; if 100l inoculum, divide by 0.10 ml]. 96-Well Titration Assay/Form BJNewton 05/08/2002 96-Well Virus Titration Assay Sample ID A 1:5 B 1:25 Volume of Inoculum: _____l 96-Well Titration Assay/Form BJNewton C 1:125 D 1:625 E 1:3125 F 1:15625 Date of Assay: ___________________ G 1:78125 H 1:390625 Titer in pfu/ml Titer = # Infectious Units (plaques or cells) X inverse of dilution / volume of inoculum [if 50l inoculum, divide by 0.05 ml; if 100l inoculum, divide by 0.10 ml] 05/08/2002, Rev. 8/24/04