TITLE: Fluorescence Titering Assay for Lentivirus
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for running a fluorescence titering assay for lentivirus. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/fluorescence-titering-assay/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bkyww.png" /></div></div>

[STEPS]
?. [Seeding cells]
Seed 75,000 cells into each well of a 6-well dish.
?. [Seeding cells]
Dilute 525,000 cells into  of DMEM complete.
14 ml
?. [Seeding cells]
Mix well by pipetting or inverting.
?. [Seeding cells]
Aliquot  of cell suspension into each well of the 6-well dish.
2 ml
?. [Seeding cells]
Incubate the cells overnight.
?. [Viral Titering]
If using freshly collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris.
Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing and aliquoting virus, it is recommended that you titer from the frozen stock to account for any loss in titer associated with freeze-thaw.
?. [Viral Titering]
If using frozen virus, rapidly thaw the lentiviral aliquot at  by agitating in a warm water or bead bath.
37 °C
?. [Viral Titering]
Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene.   ABCD1DilutionVolume of Lentivirus Stock (μL)Volume of DMEM complete (μL)Volume of 10mg/mL polybrene (μL)21:101501348.51.531:25601438.51.541:50301468.51.551:75201478.51.561:100151483.51.5
Note, this protocol was developed using low titer lentiviral vectors (105TU/mL). If you anticipate that your viral stock will be higher titer consider additional dilutions.
ABCD1DilutionVolume of Lentivirus Stock (μL)Volume of DMEM complete (μL)Volume of 10mg/mL polybrene (μL)21:101501348.51.531:25601438.51.541:50301468.51.551:75201478.51.561:100151483.51.5
?. [Viral Titering]
Gently aspirate media from the cells.
?. [Viral Titering]
Add  of a viral dilution to each well (each well gets one dilution with one well left over).
1.5 ml
?. [Viral Titering]
Count the cells in the remaining well, a cell count is required for calculating the titer.
?. [Viral Titering]
Incubate for  - .
?. [Viral Titering]
Gently aspirate media and replace with  of PBS.
1 ml
?. [Analysis]
Calculate the percent of fluorescent-positive cells in each well.
When calculating titer, only consider wells with less than 40% fluorescent-positive cells. Titering methods assume 1 integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple integration events leading to underestimation of the true titer.
?. [Analysis]
Calculate the transduction units per mL (TU/mL) using either the dilution factors (method 1) or the volume of virus (method 2):
?. [Analysis]
Method 1: TU/mL = (Number of cells transduced x Percent fluorescent x Dilution Factor)/(Transduction Volume in mL)
Method 1 example: If the 1:100 well has 25% fluorescent cells and 150,000 cells were originally transduced, then there are(150,000 x 0.25 x 100)/(1.5 mL) = 2.5x106TU/mL
?. [Analysis]
Method 2: TU/mL = (Number of cells transduced x Percent fluorescent)/(Virus volume in mL)
Method 2 example: If 15 µL of virus added to 150,000 cells resulted in 25% fluorescent cells, then there are(150,000 x 0.25)/(0.015 mL) = 2.5x106TU/mL
?. [Analysis]
For a more accurate titer, take the average of multiple dilutions.