I have sorted 8000 cells into 2mL of media and then transferred them into wells for further processing but my cells have not been able to grow
Hello Kerishnee Naicker
The cells may be under stress after sorting. During sorting you need to ensure that cells are not subjected to any kind of stressful condition. Therefore, you need to consider the following.
1. 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.
2. The collection tube also matters. 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.
3. Try to run cells at lower pressure as this will decrease the shear forces that cells experience thereby preventing the detrimental effects on the cells’ biology. Keep the sample pressure low to ensure that the core stream, through which the cells flow, is narrow and that its velocity is consistent. Another important factor to consider is the selection of an appropriate nozzle size for the cell size to be sorted. The cell sorters come with a variety of nozzle sizes with specific pressure ranges. If you are sorting larger cells, you will need a wider nozzle. You need to know that inappropriate nozzle sizes can increase cell stress and decrease viability by causing inappropriate deposition and intense shear force.
4. 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.
If you optimize the following namely, the pressure, temperature, and components of your buffer solution, the cells may be less stressed, and you may succeed in culturing them after sorting for further processing.
Best.
Thank you Malcolm Nobre
My cells were sorted at 4°C which could've been the issue
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