I tried to sort human neural stem cells (induced from hiPSC) and observed that almost all of the cells die after the sort, although they were alive before. I used 100um nozzle, and PBS-EB (EDTA and 2% final BSA). And when I tried to isolate gDNA from the sorted cells, I could not obtain any, although there should have been 0.5M cells.
Any ideas what would be stressing and killing them? Any suggestions to optimize it?
Hi Ceren,
Just going through the flow cytometer/sorter is stressful to any cell type not just neural stem cells. I would recommend sorting the cells into media with a higher percentage of FBS. The higher concentration of FBS should help them recover. For instance, if the cell type you are working with normally grows in 10% FBS you would sort them into media with 20% FBS to let them recover and then you can switch them back to 10% after a period of time. I am not sure what concentration of FBS is used with neural stem cells so my guess would be to double the concentration in the media you sort into.
Good Luck!
If you have a cell surface marker I would recommend trying to sort the cell using MACS. This causes less stress on the cells as there is no depressurisation and can be used to sort a large number of cells in a short period of time negating any time effects.
Go to the Miltenyi website to see what markers are available;
www.miltenyibiotec.com
Thanks for the recommendations. Unfortunately there is no single surface marker specific to NSC, therefore I had to use intracellular markers.
I will try the sorting again under milder conditions, as the last time I infected my cells before sorting, which might have caused extra stress on them (infection rate was about 75%)
try reducing the sorting pressure. We find with a 100um nozzle the maximum system pressure is 28 PSI, but often 12PSI yield better post sort viability.
Thanks for all the suggestions. I could not get a live population after sorting under mild conditions, and instead continued with the whole population.
Hi Ceren,
I have just started FACS and so I am not an expert but I am not sure whether you can get live cells sorted after intracellular staining. On the other hand, you might be interested to see the paper of Yuan et al 2011 (I am attaching herewith) for cell surface marker of NSC cells derived from hESCs.. Additionally you can think about using antibody against the extra cellular marker A2B5 which expresses neural stem cells.. I saw MACS do have microbeads against them as well, or you can do your FACS sorting using this antibody after extra cellular staining. Good Luck :)
Thanks, Goutam! I did see this paper before but my problem was not only with the staining. For sorting I was using a fluorescent marker (RFP), from the viral infection I do before the sort, and also wanted to sort for NSC markers. Since cells did not survive the sort with the staining, I tried sorting only for the RFP marker and still under the mildest conditions, they did not survive (100um nozzle, etc.).
I know there are papers/groups who could sort NSC and further keep them in culture but somehow in my hands under similar conditions the cells cannot make it. Thanks for the suggestions though! :)
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