cell sorting - losing cells? - (May/31/2010 )
hi everyone,
we wanna do qpcr with lymphocytes isolated from cat blood. we isolate pbmc by density gradient centrifugation on ficoll and label them with mab. our technician sorts them into 3 different populations (using the FACSaria) and we extract rna right after sorting. we have noticed that we yield very low rna concentrations which do not correlate with the number of cells we get from the sorting data. so we started counting the cells under the microscope after sorting and it turns out that the number of cells we actually get from the sort is only about 1/10 of that from the sorting data. our technician does not have an explanation for this. has anyone had similar problems? we are aware of the fact that the sorting is pretty harsh on the cells. but even if we add the number of dead cells to the number of vital cells, we are still far off the data the aria gives us.
any ideas on that would be much appreciated!
thanks!
Could you partly be sorting labelled debris?
NemaToStella on May 31 2010, 09:39 AM said:
might be. but should debris not be excluded when we set our gate on the lymphocytes? also, this would still not explain this enormous cell loss in my opinion. we start with approx. 10^8 pbmc, sort into cd4-, cd4+ and cd4+cd25+ populations, and end up with an entire number of 10^5 cells at the best.
aachleit on Mon May 31 17:33:22 2010 said:
hi everyone,
we wanna do qpcr with lymphocytes isolated from cat blood. we isolate pbmc by density gradient centrifugation on ficoll and label them with mab. our technician sorts them into 3 different populations (using the FACSaria) and we extract rna right after sorting. we have noticed that we yield very low rna concentrations which do not correlate with the number of cells we get from the sorting data. so we started counting the cells under the microscope after sorting and it turns out that the number of cells we actually get from the sort is only about 1/10 of that from the sorting data. our technician does not have an explanation for this. has anyone had similar problems? we are aware of the fact that the sorting is pretty harsh on the cells. but even if we add the number of dead cells to the number of vital cells, we are still far off the data the aria gives us.
any ideas on that would be much appreciated!
thanks!
I have been sorting for 20 years and have never the machine number match the true output. What I will
tell you is that postprocessing of the specimens are very important and it takes some experimentation
to get it right.