Determining the titer efficiency of your cells using the fluorescence marker is convenient. However, for most screening cases, it is practical to select using the antibiotic gene for transduced cells (based on the selection marker on the library vector). In this way, you get rid of non-transduced cells as soon as possible after library transduction.
While this antibiotic is easily accomplished by antibiotic selection. However, during antibiotic selection, it is important to avoid using too high an antibiotic concentration for two reasons: (1) you want to avoid the killing of a sizeable fraction of transduced cells, and (2) you do not want to enrich for cells with multiple viral integrants.
Determine the optimal concentration of antibiotic for library screens.
- Based on the titer efficiency of your cells (see Determine Transduction Efficiency by Flow Cytometry or Antibiotic Selection section), set up transduction cell pools using the amount of viral particles that resulted in 20%-50% transduced cells.
- Aliquot same amount of cells in six 12-well wells (~50,000 cells /well).
- Add increasing amount of antibiotic to each well (for puromycin selection, you would typically use 0.5 ug/ml, 1 ug/ml, 2 ug/ml 4 ug/ml, 8 ug/ml). Include a no-antibiotic control well.
- Select the lowest antibiotic concentration that resulted in >90% transduced cells selection, while maintaining the fluorescence intensity of selected and unselected transduced unchanged.
In the example below, the top histogram is the cells transduced at MOI ~0.3, unselected; the middle histogram is the transduced cells selected with an antibiotic concentration which resulted in the enrichment of transduced cells to ~90%, without affecting RFP intensity; the bottom histogram is the transduced cells selected with an antibiotic concentration which resulted in the enrichment of transduced cells to ~99%, but also increased RFP intensity (at the expense of library representation).
Need more help with this?