Seb staphylococcal enterotoxin b

Mononuclear cells from blood were isolated and viably cryopreserved. Cells were thawed, rested overnight, and 2×10⁶ cells were stimulated with 2μg/ml of cognate peptides for 6h in RPMI containing 10% human serum in the presence of 5 μg/ml of Brefeldin A (Sigma-Aldrich). Non-stimulated cells, as well as cells stimulated with super antigen SEM (SEA (staphylococcal enterotoxin A) + SEB (staphylococcal enterotoxin B) (Sigma-Aldrich), were used as controls. These cells were then stained with the following surface markers for 15 min at 4°C: CD4 (clone L200-PerCP-Cy5.5, BD Biosciences), CD8 (CD8–PE–Texas Red (ECD), Cedarlane), CD95 (clone DX2-PE-Cy5, BD Biosciences), CD28 (clone CD28.2-Pacific Blue, custom made, BD Biosciences), CCR7 (clone 3D12-PE-Cy7, BD Biosciences), PD-1 (clone MIH4-FITC, BD Biosciences). Cells were fixed for 10 minutes in 100 μl 2% paraformaldehyde at room temperature (25°C). To stain cells with antibodies specific for intracellular cytokines (IFN-γ-Alexa-700, IL-2-APC, TNF-α-PE; BD Biosciences), we incubated the cells with antibodies in 0.25% saponin (Sigma-Aldrich) for 30 minutes at 25°C and analyzed them using the BD LSRII flow cytometer. Between 250,000 and 1×10⁶ events were acquired for each condition. Data were then analyzed using DIVA software (BD Biosciences) and the frequency of cytokine producing T cells is presented.