Titering Pseudo-typed Lentiviruses

-       for titering one “batch” of collected virus

Cell Seeding

1.     Seed 6 wells of a 12 well plate with 30 000 – 50 000 HeLas per well on coverslips

Transduction

1.     Prepare “transduction media” by mixing 6 µL of polybrene (1000X) in 6 mL of DMEM+10% FBS

2.     Remove media from the plate and replace with 900 µL of transduction media in each well

3.     Begin the serial dilution by adding 100 µL of virus to the first well. There is often a vial set aside labelled as 10X diluted (the starting dilution will be a 100X dilution). Mix the

contents of the well by pipetting up and down, discard the pipette tip.

4.     Using a new pipette tip take 100 µL out of the first well and add it to the second well, mix the contents of the well the same as before, discarding the pipette tip.

5.     Continue the dilution series by adding 100 µL from the second well into the third well and mixing, followed by 100 µL from the third well into the fourth well and 100 µL from

the fourth well to the fifth well. Mixing well and discarding the tips in between each well.

6.     The last well is reserved for an untransduced control.

Preparing the cells for imaging

1.     After 24-48 hours (48 hours is preferred) remove the media and wash the cells with PBS. This should be done in the biosafety cabinet.

2.     Add 1 mL of 4% PFA to each well, incubate at RT for 20 min. After this step the plate may be removed from the biosafety cab

3.      If the vector that is packaged does not have a fluorescent tag, immunostain for the target gene.

4.     Rinse wells with PBS and add 1 mL of 1:10 000 Hoechst in PBS to each well, incubate at RT for 10 minutes

5.     Rinse 1-2 times with PBS

6.     Mount on slides or image on the same day using a spaceship

Imaging

1.     Image each dilution on 40X magnification, using the blue channel for Hoechst, and the green for ZsGreen featuring viruses, take 5 images per dilution.

2.     It is common to have very few positive cells on the 10 000X dilution and no positive cells on the 100 000X and 1 000 000X dilution. Stop imaging after you image a dilution

that has 0 cells transduced.

Calculating the viral titer

-       There is an ImageJ script for counting the cells, however the thresholding it uses isn’t always appropriate

-       Add the total number of cells counted and number of positive cells need to the appropriate columns in the "Lentivirus Titrations" spreadsheet, it will calculate the titer for you

For each dilution

1.     Count all the nuclei on all five images (you can also stop at ~ 100 nuclei)

2.     Count all the ZsGreen positive cells in all five images

3.     Calculate the % positive  (# of green cells/# of nuclei X 100)

4.     Calculate the titer of each dilution

   Titer (TU/L) = (number of cells X percent of positive cells)/(volume in well X dilution factor)

5.     Average the titers for each dilution where: the percent positive is <30% and >0%.

• The average titer is the one you will use for future MOI calculations