FACS Titration of Lentivirus Protocol

Determining the functional titer of lentiviral particles

FACS Analysis of CFU

FACS (Fluorescence-Activated Cell Sorting) provides a method for sorting a mixed population of cells into two or more groups, one cell at a time, based on the specific light scattering and fluorescence of each cell. This method provides fast, objective, and quantitative recording of fluorescent signals from individual cells.

Protocol:

Note: The following protocol includes the use of polybrene to enhance transduction. If you have a concern that your cells may be sensitive to polybrene, it may be desirable to perform a sensitivity test before proceeding with FACS titration.

Day 1

Seed wells in six-well plates to achieve 30-50% confluence on Day 2. Prepare duplicates for each dilution to be tested, including controls. Incubate cells overnight under normal culture conditions.

Day 2

1. Thaw one vial of lentiviral particles on ice until ice crystals disappear; mix by gently tapping the vial several times. Store the lentiviral stock on wet ice while in use.
   
2. Prepare media containing hexadimethrine bromide (polybrene) to a final concentration of 8 µg/mL.
   NOTE: Polybrene enhances the transduction of most cell types. Some cells are sensitive to polybrene and for some, it does not enhance transduction. Include controls for +/- polybrene to determine whether it should be used with the cell line being transduced.
   
3. Prepare 2 mL ten-fold serial dilutions of the lentivirus preparation from 1 x 10^-1 to 1 x 10^-5 in polybrene-containing complete media (if needed), using the table below as a guide. At each step, mix well to ensure homogenous mixture in each tube and change pipet tips between dilutions.

Tube Number	Dilution Factor	Virus Volume	Polybrene-Containing Complete Media Volume
1	1x10-2	22 µL	2178 µL
2	1x10-3	220 µL of Tube #1	1980 µL
3	1x10-4	220 µL of Tube #2	1980 µL
4	1x10-5	220 µL of Tube #3	1980 µL
5	1x10-6	220 µL of Tube #4	1980 µL

4. Remove medium from all wells.
5. Add 1 mL of lentivirus dilution to wells of duplicate plates, leaving one well with media alone to serve as a negative, non-transduced control. Return cells to the incubator.

Day 3

1. Remove the medium containing lentiviral particles from all wells.
   
2. Add 2 mL fresh medium (without polybrene) to each well.
   
3. Return cells to the incubator.

Day 4-6

Monitor cells under the microscope for expression of the fluorescent reporter gene. Once fluorescent cells are visible, process cells for flow cytometry.

1. Remove media and gently wash cells with 3 mL Dulbecco’s PBS. Aspirate wash.
   
2. Detach cells by adding 0.5 mL of trypsin/EDTA to each well, then incubate for 1 minute at 37 °C.
   
3. Add 1.5 mL medium to each well to inactivate trypsin/EDTA; mix well to re-suspend cells.
   
4. Transfer 300 μL of cells to 5 mL FACS tube.
   
5. Analyze cells for GFP expression using FACS.

Analysis

When calculating functional titer, choose 1% to 20% fluorophore-positive (FP-positive) populations. Below 1% FP-positive, FACS may not reliably determine the number of positive cells; above 20% FP-positive, the chance for each positive target cell to be transduced twice increases significantly, resulting in an underestimation of the number of transducing particles. Based on the chosen populations, calculate functional titer, in transducing units per mL, using the following equation:

TU/mL = (initial cell count * % GFP positive)/dilution

Now that we know the functional titer of the lentivirus particles as it correlates to the p24 ELISA titer, we can establish an accurate MOI (Multiplicity of Infection) and proceed with transduction.