Annexin 5 and propidium iodide staining

Samples of isolated platelets and PBMCs were analysed for purity after each experiment. Cells were blocked with 1:10 human Fc blocking reagent for 10 minutes at room temperature (RT) before samples were stained with fluorochrome-conjugated monoclonal anti-human Ig antibodies against CD41a and CD45 for 30 minutes in the dark. Cells were washed and resuspended in flow cytometry buffer (1% FCS in PBS) for analysis. Compensation beads (OneComp eBeads) and isotype controls were prepared in the same way. Cell viability was confirmed using by Annexin V and propidium iodide staining (BD Biosciences, Heidelberg, Germany). Flow cytometry was performed with a FACS Canto A flow cytometer and data were analysed using FACS Diva software 6.0 (BD Biosciences, Heidelberg, Germany) and the FlowJo software (Tree Star). The applied gating strategy was based on doublet discrimination and isotype-matched control antibodies.

Mononuclear cells isolated from colonic-LP were stimulated with PMA (1 µg/ml, Sigma) and ionomycin (1 µg/ml) for 6.5 h in vitro in the presence of Brefeldin A (10 µg/ml, Sigma) for the final 5 h. The cells were then harvested and surface-labeled with anti-CD4 antibody before fixation/permeabilization using BD Cytofix/Cytoperm™ kits (BD Bioscience). Intracellular staining was performed for 30 min at 4^°C using anti-IL-2 (JES5H4), IL-17 (TC11-18H10.1), IL-10 (JES5-16E3), IL-4 (BVD6-24G2), and TGFβ1 (TW7-16B4) antibodies, all from Biolegend.
For apoptosis experiments with sorted cells, DAPI⁻CD45⁺CD4⁺ T cells from colonic LP were sorted and seeded into 96-well plates for stimulation with plate-bound anti-CD3ε (clone 2C11, 5 µg/ml, eBioscience) and soluble anti-CD28 (clone 37.51, 2 µg/ml, eBioscience) antibodies in complete RPMI medium and incubated for 24 h at 37^°C. After incubation, cell death was quantified by Annexin V and propidium iodide staining (BD Bioscience).

Cells undergoing apoptosis were identified by annexin V and propidium iodide staining (BD Pharmingen, USA) according to the manufacturer’s instructions. Briefly, the cells were washed twice with cold PBS and then were resuspended in 1X binding buffer at a concentration of 1×10⁶cells/ml. Then, 100 μl of the solution (1×10⁵ cells) was transferred to a 5 ml culture tube. 5 μl of annexin V-FITC and 5 μl of PI were also added. Then, the cells were vortexed gently and incubated for 15 min at RT (25°C) in the dark. Finally, 400 μl of 1×binding buffer was added to each tube and they were analyzed using FACSCalibur flow cytometer (Becton-Dickenson, Mountain View, CA, USA) and FlowJo software.