Lentivirus titer quantification by flow cytometry (FCM)

As the 12 K lentiviral vector expresses an EGFP gene, the functional titer of our lentivirus prep was assayed by FCM. Briefly, (1) split and seed HEK293T cells to 24-well plate on day 1, 5 × 10⁴ cells per well. Generally, 18 wells were used to perform the titter detection, a gradient volume of the crude lentivirus was added into the cells and each volume was tested by replicates. In this experiment, the crude virus gradients were 10, 20, 40, 80, and 160 µl for each well (Supplementary Fig. ⁵). Another two wells of cells were used for cell counting before transduction; (2) Conduct lentivirus transduction when cells reach up to 60–80% confluence on day 2. Before transduction, detach the last two wells of cells using 0.05% EDTA-Trypsin to determine the total number of cells in one well (N_initial). Then change the remaining wells with fresh culture medium containing 8 µg/mL polybrene, then add the gradient volume of crude virus into each well and swirling gently to mix; (3) On day 3, change to fresh medium without polybrene; (4) On day 4, harvest all the cells and wash them twice in PBS. Fix the cells in 4% formalin solution at RT for 20 min, then spin down the cell pellet at 500 × g for 5 min. Discard the supernatant and re-suspend the cell pellet carefully in 600 µl PBS, and conduct FCM analysis immediately. FCM was performed using a BD LSRFortessa^TM cell analyzer with at least 30,000 events collected for each sample in replicates.

The FCM output data was analyzed by the software Flowjo vX.0.7. Percentage of GFP-positive cells was calculated as: Y% = N_{GFP-positive cells}/N_{total cells} × 100%. Calculate the GFP percentage of all samples. For accurate titter determination, there should be a linear relationship between the GFP-positive percentages and crude volume. The titter (Transducing Units (TU/mL) calculation according to this formula: TU/mL = (N_initial × Y% × 1000)/V. V represents the crude volume (µl) used for initial transduction.