Could anyone give me a suggestion how to isolate Tregs by magnetic separation? I need to use Tregs for cultivation then, so I suppose negative selection is necessary. I wanted to use CD25+ CD49d- selection, but in the second step here the positive selection is used, so I don´t know.
Probably easiest to a CD4+CD25+ regulatory T cell enrichment (using whichever companies beads you wish) first. Then use MACS fluorochrome beads. Label your cells with CD49d (either FITC, PE or APC) and use the respective bead to purify on a MACS column. Collect the negative cells for your CD49d- cells... Hope that helps..
Adnan, if I do CD4+25+ enrichment first with magnetic beads, these cells will keep magnetic particals on the surface ....and even if the next stem is negative, Tregs won´t be untouched in the end. Maybe I´m wrong? The problem is we don´t have a sorter yet, then everything would be easier....that´ s why I´m looking for a magnetic separation at the moment.
Now I have found another isolation kit for CD4+CD127lowCD49d- cells, which should leave the cells untouched, it is not based on CD25 expression, but still it could work I hope
That isolation kit should work... If you wish to use CD25 as a means for selecting Treg then you can do a negative selection for CD4 cells then use the fluorochrome approach to select CD49d negative cells first followed by selection of CD25..
Look here for clinical scale Tregs
http://www.ncbi.nlm.nih.gov/pubmed/23772685
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