Human cd4 cd25 treg isolation kit

PBMCs were isolated from healthy fertile women as described previously.^{35 (link)} Naïve CD4⁺ T cells, CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T cells were purified by MACS using the human naïve CD4⁺ T Cell Isolation Kit and the human CD4⁺CD25⁺ Treg Isolation Kit, respectively, according to the manufacturer's instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of the separated naïve CD4⁺ T cells, CD4⁺CD25⁺ T cells and CD4⁺CD25⁻ T cells was >95%, as determined by flow cytometry.

To determine whether the Treg from MMD have immunosuppressive capacity, the proliferation effect of T cells was measured by flow cytometry. Treg cells were isolated using human CD4⁺CD25⁺Treg Isolation Kit (MiltenyiBiotec, Inc., Auburn, CA) according to the manufacturer’s instructions. The CD4⁺CD25⁻ fraction was defined as effector T (Teff) cells. The Treg suppressive assay was conducted as described before^{15 (link)}. In brief, 5 * 10⁴ carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled Teff cells (2.5 µM) per well were plated and cultured in a 24-well plate with Treg cells (Teff/Treg = 1:1) in the presence of CD3/CD28 antibodies (5 ng/ml), TGF-β (10 ng/ml) and IL-2 (100 IU/ml) for 96 h. The Teff cells were harvested and evaluated by flow cytometry. Cultured CD4⁺CD25⁻ cells were taken as control group. The data were analyzed using Modfit software (Verity Software House 2.0, Top-sham, ME, USA).

Buffy coats of healthy blood donors were received from the Institute of Clinical Transfusion Medicine, Ulm, Germany. All experiments were approved and followed ethical standards and local regulations. PBMC were purified by Ficoll gradient centrifugation. Treg (CD4⁺CD25⁺) and conventional T cells (Tcon; CD4⁺CD25⁻) were isolated by a human CD4⁺CD25⁺ Treg isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, CD4⁺ T cells were isolated by negative selection, followed by positive selection of CD25⁺ cells (Treg). The remaining CD4⁺CD25⁻ fraction was used as Tcon. Allogeneic APC were prepared by depletion of CD3⁺ cells from PBMC of a different donor using CD3 microbeads (Miltenyi Biotec). For some experiments, untouched CD3⁺ pan-T cells were isolated (EasySep human T cell isolation kit, STEMCELL Technologies).