Download 24 Hour Fluorescent Virus Titration Assay (96

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 (80l clear DMEM/7.5% FCS + 20l
virus) as described below:
FIRST, using an 8-channel pipettor, put 80l clear DMEM/7.5% FCS into each well of the blank 96-well plate(s). Add 20l
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 20l diluted virus from Columns 1-6, Row A to Row B.
CHANGE TIPS!
Mix by pipetting up and down 8-10 times, then transfer 20l diluted virus from Row B to Row C. CHANGE TIPS! Continue
down the plate until row H is mixed. Change tips after dropping each 20l 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 50l 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
50l inoculum, divide by 0.05 ml; if 100l 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 50l inoculum, divide by 0.05 ml; if 100l inoculum, divide by 0.10 ml]
05/08/2002, Rev. 8/24/04