Hi, guys:
I am sorting Tregs cells from the spleen and LNs of Foxp3-GFP KI mice now. In fact, I met some problems recent: first, the CD4+ Foxp3+ Cells are gated above lymphocytes population. Second, it is recorded that I collected 10 0000 cells in Sorting layout but I cannot find cells under microscope. May I get some information about efficient sorting, both from Abs staining and drop layout? Thanks a lot.
Hi Shuoyang,
this question is very hard to answer without actually being there at the sorter. I dont quite know what you mean by by saying that the CD4+ Foxp3+ cells are "above the lymphocyte population"... if you say that you sorted 100.000 cells but cant find them under the microscope, there can be a variety of reasons, the most obvious being that the side streams were not properly aligned into your collection tube, or that you were sorting into a dry tube. If you have not done so, I would take the following steps/precautions:
- Pre-wet your collection tube either with medium with FCS or pure FCS.
- Have your sorting operator make sure that the drop delay is set properly, and that the deflected side stream is hitting your collection tube.
- Use a singlet gate and a live/dead marker
- Reanalyze your cells at the sorter after your sort (i.e. aquire a bit from your collection tube).
There is no way that the sorting layout at the sorter will tell you what happened to your cells, it will only tell you how many of your cells have been deflected, what the used sorting mask was, and some other technical details.
Good luck with your repeat experiment.
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