5.3. Determine Transduction Efficiency by Flow Cytometry or Antibiotic Selection

For lentiviral constructs with a fluorescent marker or antibiotic resistance marker, transduction efficiency (i.e., % infected cells) can be determined from the fraction of fluorescent or antibiotic resistant cells in the population. To calculate the viral titer, it is also important to know the total number of cells at the time of transduction.

To determine viral titers, transductions should be set up as a serial dilution range of different amounts of virus in 12-well plates, 100,000 cells/well. For example, when titering standard virus from Cellecta, we recommend aliquoting 0, 0.1, 0.3, 1, 3.3 of concentrated stock to 5 different wells (or, for non-concentrated virus, scale up volumes ca. 100-fold). If you are using cells that transduce with low efficiency (e.g., primary cells, some suspension cell lines, etc.) you may need to use 10 μl, or even 30ul, of concentrated virus stock for titering. Always be sure to include the initial well of cells only (no virus) as a negative control.

Option 1: Use flow cytometry with a fluorescent protein marker to determine % infected cells:

IMPORTANT: Do not use fluorescence microscopy to assess the percentage of transduced cells. A significant number of transduced cells will be missed, so transduction efficiency will be underestimated.

1. About 72 hours after adding virus to the cells, spin down and resuspend cells in plate in 1X D-PBS.

2. If trypsin is used, block it with FBS/media, then centrifuge.

3. Determine the percentage of transduced (RFP/GFP/BFP-positive) cells by flow cytometry. For detection of Cellecta’s RFP-, GFP-, and BFP-positive cells use the following settings:
   

   Flow Cytometry Settings for TagRFP
   Excitation:	561nm (530nm laser is still acceptable)
   Emission:	600/20nm band-pass filter, or similar
   Flow Cytometry Settings for TagGFP2
   Excitation:	488nm
   Emission:	530/20nm band-pass filter, or similar
   Flow Cytometry Settings for TagBFP
   Excitation:	405nm (violet laser)
   Emission:	460/20nm band-pass filter, or similar

4. Use the formula below to calculate the percentage of transduced cells:

(Fluorescent cells / Total number of cells) x 100 = % infected cells (use this for the titer calculation)

Option 2: Use antibiotic selection to determine % infected cells:

1. About 72 hours after adding virus to cells following the standard transduction protocol, split each transduction dilution (including the no-virus control) into 2 separate twin wells using 1:8 split ratio.

2. Add an appropriate amount of antibiotic to one twin well for each viral dilution (including the no-virus control).

3. Incubate all cultures for 2-5 additional days to allow antibiotic time to kill >99% cells in the no-virus control. Do not let the cultures reach confluence (split if needed).

4. After the antibiotic has had time to kill the cells, remove media from cells and replace with fresh media containing a 1/10 total volume alamarBlue stock solution. Include a media-only sample (no cells) for a background alamarBlue reading.

5. Incubate 1 hour at 37°C. Read the fluorescence intensity of the alamarBlue staining using a plate reader. Use the alamarBlue fluorescence values in the following calculation to determine the percent transduced cells in each antibiotic/non-antibiotic pair:

( [antibiotic-selected cells – media-only] / [non-selected {no antibiotic} cells – media-only] ) x 100 = % infected cells (use for titer calculation)