TITLE: Colony Formation 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 colony formation titering assay for Lentivirus. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/colony-formation-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/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqjs7esn.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/5acd19e056bdf88808866a63dd76bebaf3bfacef4acb2f29cfb4ece3805005bf/bqjt7esn.png" /></div></div>

[STEPS]
?. [Procedure]
Before beginning a colony formation assay, the dose of antibiotic required to kill your target cell line needs to be empirically determined. Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration required to kill all of the cells. Use this dose for the colony formation assay.
?. [Procedure]
Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting  of 10 mg/mL polybrene into  media.
20 µl
20 ml
?. [Procedure]
If using freshly collected virus, filter through a 0.45 μm polyethersulfone filter to remove cells and debris.
?. [Procedure]
If using frozen virus, rapidly thaw the lentiviral aliquot at  by agitating in a warm water or bead bath.
37 °C
?. [Procedure]
Prepare 10-fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene:
Notes:Mix the dilutions wellThe 1:10 dilution can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately quantified.
?. [Procedure]
Add 150 μL of a viral dilution to each well (each well gets one dilution)
?. [Procedure]
Seed 1,000 cells into each well of a 6-well dish.Prepare a batch of cells as follows: Dilute 7,000 cells into  of DMEM complete containing 10 μg/mL polybrene.Aliquot  of the cell suspension into each well of the 6-well dish directly on top of virus. Swirl gently to mix and place dish in incubator.
9.45 ml
1.35 ml
?. [Procedure]
Incubate for -.
*Pro-Tip*Different cells may be more sensitive to lentivirus or to polybrene, in which case this incubation step can be reduced to   and the media can be replaced with DMEM complete. However, antibiotic-containing media should not be added until  -  after transduction.
?. [Procedure]
Gently aspirate the media from the cells.
?. [Procedure]
Add  of DMEM complete containing the appropriate antibiotic.
1.5 ml
?. [Procedure]
Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative) control well should be killed and colonies should be visible.
?. [Procedure]
Every 3-4 days, gently aspirate the media and replace it with fresh DMEM complete containing the appropriate antibiotic.
?. [Procedure]
When the untransduced (negative control) cells have died and colonies are visible in the transduced wells, aspirate the media from the wells.
?. [Procedure]
Gently wash the cells with  of PBS and aspirate wash.
1 ml
?. [Procedure]
Filter 0.1% crystal violet solution through a 0.22 μm filter to remove any precipitates.
?. [Procedure]
Stain each well with  of 0.1% crystal violet for  at room temperature.
1 ml
?. [Procedure]
Gently remove the stain.
?. [Procedure]
Wash cells 3x with  of PBS, being careful not to disturb the colonies.
1 ml
?. [Procedure]
Count the colonies for at least 2 of the dilutions.
*Pro-Tip*The higher dilution wells tend to provide more accurate titers as the likelihood of multiple integration events decreases as the dilution increases.
?. [Procedure]
Calculate the transduction units per mL (TU/mL) as follows:e.g., If the 1:100,000 well has 75 colonies, then there are 75 colonies / x 1,000,000 or 50,000,000 TU/mL
1.5 ml
?. [Procedure]
For a more accurate titer, take the average of multiple dilutions.