Facs aria 1 sorter

Peripheral blood mononuclear cells (PBMCs) were isolated from blood of patients or from the buffy coat preparations of healthy donors by standard Ficoll-Paque™ Plus density-gradient centrifugation (GE Healthcare). PBMCs were labeled with anti-CD4, anti-CD127, anti-CD25, and anti-CD45RA monoclonal antibodies, incubated at 4°C in the dark for 20 min. For intracellular staining, cells were permeabilized with 150 μL of fixation/permeabilization solution at 4°C for 12 h. After two washes with permeabilization buffer 1×, PBMCs were incubated with anti-FOXP3 and anti-Helios for 30 min at 4°C in the dark. Samples were acquired with an Attune^® Acoustic Focusing Cytometer (Life Technologies) and FlowJo 7.6.2 software (Tree Star, Ashland, OR, USA) was used for data analysis. A total of 100,000 events were acquired for analysis, after gating of lymphocytes based on the FSC/SSC dot plot.
For sorting, 50 × 10⁶ PBMCs were washed once and resuspended in media (RPMI/10% FBS) at 100 × 10⁶ per ml. Cells were incubated with anti-CD4 and anti-CD25 at 4°C for 30 min, washed twice and resuspended in PBS 1×. Labeled cells were sorted using a FACs Aria I sorter (BD Biosciences). For Treg isolation, a CD4⁺CD25^veryhi gate was used and sorted cells were collected in media (RPMI/20% FBS), washed once and resuspended in culture media until ready to be plated in the suppression assay.

Tregs were identified as CD4+/CD25+/FoxP3 positive cells by flow cytometry analysis. The staining was performed using Treg detection human kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and the results analyzed by BD CANTO II flow cytometer and DIVA software (Becton-Dickinson, Franklin Lakes, New Jersey, USA). A total of 100,000 events were acquired for analysis after gating of lymphocytes based on the FSC/SSC dot plot. For sorting, labeled cells were sorted using a FACs Aria I sorter (BD Biosciences). For Treg isolation, a CD4+CD25^veryhi gate was used, and sorted cells were collected in media (RPMI/20% FBS), washed once, and suspended in culture media.

Peripheral venous blood samples were collected in heparin-containing tubes, and PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus solution (Amersham Biosciences, Uppsala, Sweden). NK cells were phenotypically identified as CD3-CD56+ cells by flow cytometry, as previously described [21 (link),22 (link)]. NK cells were isolated using CD56 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD3-CD56+ cells was greater than 95% as analyzed by flow cytometry. For sorting of CD69+ and CD69- NK cells, PBMCs were stained with APC-conjugated anti-CD3, FITC-conjugated anti-CD56, PerCP-conjugated anti-CD45, and PE-conjugated anti-CD69 mAb, and were sorted to obtain CD45+CD3-CD56+CD69+ and CD45+CD3-CD56+CD69- NK cells using a FACS Aria I sorter (BD Biosciences, Mountain View, CA) at purities of > 98%.