Hey guys, recently I've been having some problem with cell sorting.
I have a pool of cells that contains GFP and mCherry positive HeLa cells (by saying positive I mean they have strong green and red signal under a microscope).
To get the cells that have strong GFP and mCherry signal, I scheduled a cell sorting at the platform of our school, and used wild type HeLa cells as a negative control for gating. According to the results, the sorted cells belongs to a cluster that is quite different from wild type HeLa cells, they have an obviously higher GFP-A and mCherry-A for GFP and mCherry.
However, after sorting, there were even less fluorescent cells under a microscope.
Should I also set up a group of cells that are GFP and mCherry under a microscope as a positive control? The technicians at the platform set the parameters for cells sorting and I am not quite familiar with the process. Has anyone met this problem before and can answer this question?
Theoretically - negative control should suffice if you are sure your sample is positive. If you are not sure your sample is positive, however, it is necessary to have a positive control.
But if I understand correctly - you do see florescence in the microscope, so maybe the gating at the sorting stage is not a problem. It is important to understand that sorting is a very traumatic procedure for the cells, so it is possible that your cells are just dying due to the sorting, and loose their fluorescence. I would try sorting a larger amount of cells and sorting into a serum rich media. If possible, allow the cells a few hours and up to a few days to recover. apart from this, and this depends on your technician and machine - maybe try cleaning the machine, to negate the option of contamination, and maybe working with lower speed and pressure to prevent physical strain on cells.
You mean while collecting positive cells that contains GFP and mCherry positive HeLa cells
You use Hela cells as a negative negative control for gating. (I will suggest use unstained of the same sample to make a gating). Probably there is the problem in gating and you may loss the best cells. Gating position is very important if you are interested in collecting some specific population.
Try to use unstained and single stained to make proper gating and you will know the real position of your required cells population and then make a gate on the basis of that.
Also sometimes there is problem in machine if its public( means whole institute is using), in such case try to run the wash for little long time
Hi Chen,
Keeping controls (both positive and negative) is important for setting up sort gate. How many cells did you sort, it was not clear. Additionally, sort stress can cause quenching of fluorescence for a while. You can use serum coated tubes containing your media of interest to collect the cells and make sure you are maintaining them on ice while collecting.
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