Cd4 cd25 cd127dim regulatory t cells isolation kit 2

Peripheral blood mononuclear cells (PBMC) from healthy volunteers were isolated by centrifugation using Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden). Isolated cells were washed twice with PBS and used for the negative immunomagnetic selection of untouched CD3⁺ T cells using the Pan T cell isolation Kit, according to the manufacturer’s recommendations (MiltenyiBiotec, Bergisch Gladbach, Germany). In brief, all blood cells (except CD3⁺ T cells) were depleted from PBMC using a mixture of antibodies (anti-CD14, CD16, CD19, CD36, CD56, CD123 and Glycophorin A) linked to magnetic microbeads, using magnetic columns and a QuadroMACS magnet (MiltenyiBiotec). The CD4⁺CD25⁺CD127^dim/– Regulatory T Cells Isolation Kit II (MiltenyiBiotec) was used for nTregs isolation. Briefly, PBMCs were incubated with a cocktail of biotinylated antibodies (anti-CD8, CD19, CD123 and CD127) and Anti-Biotin MicroBeads for the depletion of non-CD4⁺ and CD127^high cells. After the negative selection, the pre-enriched CD4⁺CD127^dim/− T cells were incubated with CD25 MicroBeads for subsequent positive selection of CD4⁺CD25⁺CD^127dim/− regulatory T cells. All separations resulted in purities above 95%, as determined by flow cytometry (anti-CD3 FITC for T cells and anti-CD4 FITC, Anti-CD25 PE and Anti-CD127 APC for nTregs).

PBMCs were isolated from buffy coats of healthy donors by density gradient centrifugation using Lymphoprep™ solution (07801, STEMCELL Technologies Inc.,Vancouver, BC, Canada). CD14⁺ monocytes, CD4⁺ T cells, and CD4⁺CD25⁺CD127^dim/− Tregs were separated with magnetic-based isolation kits using the autoMACS Pro Separator station (Miltenyi Biotec, Bergish Gladbach, Germany) according to manufacturer’s protocol.
CD14⁺ MicroBeads (130-096-533, Miltenyi Biotec, Bergish Gladbach, Germany) were used to separate CD14⁺ monocytes via positive selection, CD4⁺ T cell Isolation Kit (CD4⁺ T cell Microbead Cocktail, CD4⁺ T cell Biotin-Antibody Cocktail, human, Miltenyi Biotec, Bergish Gladbach, Germany) to separate CD4⁺ T cells via negative selection and CD4⁺CD25⁺CD127^dim/− regulatory T cells isolation kit II (human, 130-094-775, Miltenyi Biotec, Bergish Gladbach, Germany) to separate Tregs via pre-enrichment of CD4⁺CD127^dim/− and subsequent positive selection of CD4⁺CD25⁺CD127^dim/−.