PBMC-MSC Co-cultures for Induction of Treg Generation

Materials and Reagents

1. Peripheral blood mononuclear cells (PBMC) [isolated from a healthy donor using Ficoll-Paque (own pharmacy) density gradient (1.077 g/cm³)]
   
2. Multipotent stromal cells (MSC) from healthy donors
   
3. Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
   
4. Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
   
5. L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
   
6. Fetal calf serum (FCS) (Greiner Bio-One GmbH)
   
7. Phosphate buffered saline (PBS)
   
8. Trypsin/EDTA (1x 0.05% Trypsin-EDTA, phenol red) (Life Technologies, catalog number: 25300-096 )
   

Equipment

1. T75 culture flasks
   
2. 12-well transwell plates (pore size 0.4 μM) (Sigma-Aldrich, catalog number: CLS3460 )
   
3. 10 K Centriprep Centrifugal filters (Millipore, catalog number: 4304 )
   
4. 12/24/48-well plates (Sigma-Aldrich, Corning Costar cell culture plates)
   
5. 37 °C, 5% CO₂ cell culture incubator
   
6. Microscope
   
7. Centrifuge
   
8. 10 K Centriprep centrifugal filters
   
9. Hemocytometer (counting chamber) or Sysmex F-820
   

Procedure