PI dyed cells could be dying due to the stress and mechanical damage caused by the FACS process itself. PI only stains cells with compromised membranes, so if they were initially PI-negative, they were alive at the time of staining—but this doesn’t guarantee post-sort survival.

Hello Zhen You

I agree with Kudakwashe Mushavanhu. The FACS process itself involves high speeds, pressure, and mechanical forces, which can stress cells and potentially disrupt their membranes, even in cells that appear live. This stress might not be immediately obvious during the sorting process but can lead to cell death later during cultivation.

You may term it as “delayed cell death” meaning the cells sorted as live might have had their membranes compromised, leading to a slow decline in viability and eventually death after a week of cultivation.

You may adopt the following measures to prevent cell death after FACS sorting.

1. Prepare your sample with appropriate filtration to remove clumps and debris that can clog the nozzle. Dilute samples appropriately to avoid over-crowding the system.

2. For staining, use minimal amounts, and consider using non-toxic dyes. Avoid dyes that can be toxic, especially to fragile cells. Dyes such as PI or 7AAD are considered toxic.

3. Choose the appropriate nozzle size and pressure for your cell type. Over-pressurizing or using an inappropriate nozzle size can damage cells.

4. Your collection tubes should contain a suitable medium to gently collect cells. Impacting cells directly on the plastic of the collection tube can significantly reduce viability.

5. Minimize the sorting time. If you feel that sorting may extend, consider removing and re-suspending sorted cells at regular intervals.

6. For post-sort handling, keep collection tubes on ice until you're ready to process the cells further. This slows down metabolic processes and can help maintain viability.

7. Process the sorted cells as quickly as possible to minimize stress.

8. It is recommended that you pre-coat collection tubes with a buffer solution to mask the plastic's charge and improve cell viability, as one of the measures taken during post-sort handling.

Best,

PI is generally used at concentrations that don't immediately kill cells, prolonged exposure or slightly higher than recommended concentrations could potentially cause some level of delayed toxicity in a subset of cells that were just below the detection threshold for staining during sorting. However, this is less probable if you're using standard protocols. Instead of the PI dye you're using to tell live and dead cells apart, you could try other special dyes that work in a slightly different way. These new dyes might be gentler on the cells. For example, some of these dyes stick to dead cells because their outer layer is already damaged. However, if you try these different dyes, make sure they don't mess up the cell sorting process or the way you grow the cells afterwards.

Furthermore, the sorting buffer's components (BSA, EDTA, osmolarity, pH) could be stressing the cells. The significant environmental change from the buffer to culture medium post-sort might also be difficult for them to adapt to. Additionally, a subset of cells already in very early stages of death might appear live during sorting but succumb later in culture.