Hello Samya Dey

During FACS, if the cells are exposed to stress, the consequence is cell death. So during cell sorting, you could make some changes in your protocol in order to minimize the impact of stress on cells. You need to keep your cells happy while isolating pure population of cells.

You should make sure that you sort the cells in a medium in which the cells remain happy. Usually a medium containing high serum concentration would be appropriate (around 30-40% serum). Cell culture medium buffered with a CO2-carbonate system would not be a good choice for pH-related reasons.

The collection tube is another factor you should consider. Cells may be damaged if they hit the side of a collection tube. So ensure that the collection tube contains the appropriate medium and at the appropriate level so that the cells do not stick to the walls of the tube.

The fragility of the cells to be sorted is another significant factor. You should run cells at lower pressure as this will decrease the shear forces that cells experience thereby preventing the detrimental effects on the cells’ biology. Also, keep the sample pressure low to ensure that the core stream, through which the cells flow, is narrow and that its velocity is consistent.

The most important factor is the nozzle size. As a general rule, your cells should be no larger than one quarter the diameter of the nozzle with which you are going to sort.

Always keep your cells on ice in order to slow down the metabolic processes. But you should also know that mammalian cells become stressed and die quickly at 4°C as cold temperatures prevent cells from repairing any damage that may be caused during the sorting process. So, see to it that you choose the best temperature for your cells during sorting.

I feel that if you optimize on the above factors, especially the pressure, nozzle size and components of your buffer solution, the cells may be less stressed and you may succeed in culturing the GFP-positive cells after sorting.

Best.

Hi Samya Dey, it is normal what you got...However you can always improve your methodology, for instance, lowering the intensity of the laser.

I would give you some advices, but many are reflected on the answer of Malcolm Nobre, so I think it is enough.

To include a viability dye to your tube would ensure that you are sorting the live cells, and also as recommended by others to increase FBS or BSA concentration in your collection tube would help to keep cells in better condition.

The decreased viability and signs of perforation observed in HL60 cells after FACS sorting can be attributed to mechanical stress during sorting, apoptosis induction, photo-induced damage from lasers, and cell detachment from the culture substrate. To improve cell survival, it is recommended to optimize sorting parameters, use pre-sorting strategies to reduce the number of cells subjected to sorting, handle cells gently, optimize buffers, and provide post-sorting recovery conditions such as transferring cells to fresh media and monitoring their viability. Additionally, assessing the impact of sorting on cell phenotype and function in subsequent experiments can be helpful.