I am working with mouse T cell. I was using flow cytometry to isolate CD3+ Cd4+ and CD8+ cells from spleen. I had good results until last month when the positive cell number decreased by half. I also used CD90beads-- I got the same decreased cells from the CD90 magnetic sorted cells. I attached my result. The top one are good ones and the bottom one are bad ones.
I appreciate your help.
Beru

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Attached file: forum.jpg (37 downloads, 83KB)

It seems that the percentage of CD3,CD4 and CD90 in the bad files are more resonable than the good file.

There are many things that can be going wrong. I have to say I'm not sure what you were sorting in the top panel, because less than 50% purity of T cells by flow cytometry is not 'Good'.

Some things to consider:
Some cell-surface molecules are prone to capping, CD3 is one of them. You can prevent this by keeping the cells cold and using about 0.01-0.05% sodium azide in your buffer used to stain, wash and sort the cells.

Any problem in the setup of the FACS can lead to poor purity. For instance if your drop delay is incorrect to start with or changes during the sort, then you'll get poor purity.

Make sure the flow process is being watched. A small temporary clog can send all the wrong cells in your tubes (and affect drop delay).

As for magnetic sorting, make sure you are using the correct ratio of beads to cells.

I'm not sure how you are staining or-restaining the magnetically isolated cells, but make sure your not blocking the antibody you use for the final analysis by the presence of unconjugated antibody used to isolate the cells.

It is also worth remembering that your problems may not just be one thing and that a couple of small independent issues may be affecting your results.