After infecting Hepa1-6 cells with an empty virus vector and a specific library, the cells were resuspended in BODIPY 493/503 (HY-W090090, MCE) fluorescent dye at a working concentration of 2 µM and incubated at room temperature for 20–30 minutes with shaking to ensure sufficient staining.

After staining, the cells were centrifuged at 300 ×g for 3 minutes, and the supernatant was removed to obtain the pellet. The cells were then resuspended in 1× PBS and centrifuged again at 300 ×g for 3 minutes. The supernatant was discarded, and the pellet was resuspended in an appropriate amount of 1× PBS and placed on ice for flow cytometry sorting to obtain cell populations with enhanced or reduced fluorescence. However, during the flow cytometry sorting process, it was observed that gently shaking the cells to resuspend the sediment formed after standing caused an overall rightward shift in fluorescence intensity (i.e., an increase in fluorescence intensity), which returned to its original level after a period of time. Why does this occur, and what are the possible solutions?