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