In vitro Suppression Assay and Transmigration Assay

Protocol title: In vitro Suppression Assay 

1. Spleen, cervical and mesenteric lymph node cell suspension was obtained by passing the tissues through a cell strainer (40 μm), in PBS containing 3% fetal calf serum (FCS).

2. Erythrocytes were removed by incubating the samples with RBC Lysis Buffer (eBioscience, San Diego, CA, USA) for 5 min.

3. Then, the cells were transferred into PBS+3% FCS.

4. Approximately 60×10⁶ cells (in 600 µl) were placed in a 2 ml eppendorf tube that fits within a BD IMag™ Cell Separation Magnet (BD Biosciences) compatible with the beads that we have used. Then, 10 µl of biotin conjugated anti-mouse CD25 (eBioscience) (0.5 mg/ml) was added into the suspension of the cells, and the tube was kept on ice for 20 min, occasionally gently shaken by hand. After the indicated period, the tube was filled with 1 ml of PBS+3% FCS, centrifuged at 550 g for 3 min, washed once again with PBS+3% FCS, and finally washed with cold magnetic bead buffer (PBS+0.5% BSA+2 mM EDTA). 

5. On pelleted cells, 800 µl of cold magnetic bead buffer was added containing BD IMag™ Streptavidin Particles Plus–DM (1:20, BD Biosciences, Bedford, MA, USA). The tube was placed on ice for 30 min with occasional shaking of the tube.

6. The appropriate cells were purified by placement into the BD IMag™ Cell Separation Magnet (BD Biosciences), 3× for 8 min. Cells attached to magnetic beads can be found on the wall of the tube next to the magnet. So, by careful removing the supernatant that contains cells that are not attached, with a 1 ml pipette, the purity of the wanted CD25⁺ cells increase. Each repetition of this procedure increases the purity of cells. The supernatants should be kept on ice as they are the source of the effector CD4⁺CD25^- cells, which can be isolated second.

7. Finally, CD25⁺ cells attached to the wall of the tube (positively selected) were washed with PBS+3% FCS (2× at 550 g, for 3 min) and were finally resuspended in a T lymphocyte medium – RPMI 1640 supplemented with 10% FCS, 1% penicillin and streptomycin, 0.02 mM Na-pyruvate, 5 μM β-mercaptoethanol, 2 mM L-glutamine, and 25 mM HEPES. This way, cells which express CD25, predominantly T regulatory cells, were obtained. 

8. The procedure was then repeated with biotin conjugated anti-mouse CD4 (eBioscience) (10 µl of the stock antibody concentration of 0.5 mg/ml) on the CD25^- cells that were removed from the tubes during magnetic separation of CD25⁺ cells. Again, this is a positive selection as the selected cells contain attached beads. So, at the end of magnetic bead separation, the cells that are obtained are CD4⁺CD25^-.

9. For the suppression assay, CD4⁺CD25⁻ cells were first incubated with 2 μM CFSE (Invitrogen) for 20 min at room temperature and 5 min at 37°C, washed and resuspended in the T lymphocyte medium.

10. A U-bottom 96-well plate (Sardstedt, Numbrecht, Germany) was coated with 50 µl of anti-mouse CD3 (1 μg/ml, eBioscience) that was resuspended in PBS. The plate was kept at 37°C for 2 h, and then washed 2×with PBS.

11. Equal number of purified CD4⁺CD25⁻ cells (25×10³) was placed in each well (50 µl). CD25⁺ cells (50 µl) were then added in a series of dilutions, starting from 25×10³ cells per well, and continuing to a 2×, 4×, 8×, and 16× lesser number of cells. Certain wells contained only CD4⁺CD25⁻ cells, as control. The cells were also stimulated by the addition of anti-mouse CD28 (final concentration 1 μg/ml, eBioscience) to the T lymphocyte medium (50 µl). 

12. After 3 days of cultivation, the cells were washed, resuspended in PBS and analyzed by flow cytometry.

Protocol title: Transmigration Assay

1. First, CD25⁺ cells were isolated using the BD IMag™ Streptavidin Particles Plus–DM beads as explained in the previous protocol. 

2. For the isolation of pancreatic islets, the pancreata were first placed in a Petri dish, washed in Hanks' Balanced Salt Solution (HBSS) and the fatty tissue was removed. The pancreas was then minced with scissors into 2-3 mm long pieces, and placed into HBSS+10% FCS containing collagenase V (1 mg/ml, 3 ml per pancreas). The samples were placed in a water bath shaker and shaken for 15-20 min at 37^oC (75 RPM). Once the solution became cloudy, the digestion was stopped with cold HBSS. The samples were then left for about 15 min to settle, the solution was then removed and new HBSS was added, to a final volume of 15-20 ml. The pancreatic islets were then handpicked under a microscope. This was performed at a 40× magnification, with the light source above the Petri dish. It is best to make the bottom of the Petri dish dark (with a permanent marker, for example), for higher contrast.

3. For the transmigration assay, a special chemotaxis system was used (ChemoTX System, Neuro Probe Inc., MD, USA). This is the plate with a special membrane where the cells are placed.

4. Below the membrane of the plate, the T lymphocyte medium was placed, containing either the chemokine CXCL12 (10 ng/ml, Gibco, ThermoFisher Scientific), or the isolated syngeneic pancreatic islets (5 per well). The volume of fluid in the lower chamber was 200 µl.

5. CD25⁺ cells (1×10⁵) in 50 μl were placed on top of the membrane.

6. After 4 h at 37°C with 5% CO₂, the number of cells that migrated through the membrane and into the wells was determined by LUNA-II™ Automated Cell Counter (Logos Biosystems, Gyeonggi-do, South Korea).