Cd8 pe clone sk1

Fifteen thousand DCs were prepared as described in 2.7 and cocultured with 150,000 autologous CD3⁺ T cells at a concentration of 1 × 10⁶ T cells/ml. After 5 days of coculture, T cells were harvested, washed with PBS and stained for 15 min at the dark using the following anti-human mAbs: CD3 APC-H7 (clone SK7, BD Biosciences), CD4 Pe-Cy7 (clone SK-3, Thermofisher), CD8 PE (clone SK1, BD Biosciences), CD279/PD-1 APC (clone MIH4, Thermofisher), CD197/CCR7 Alexa Fluor 647 (clone G043H7; Biolegend), and CD45RA V500 (clone HI100; Biolegend). Unstimulated CD3⁺ T cells were used as negative control, and unstained CD3⁺ T cells were used as negative fluorescence control. At least 10,000 events of each sample were collected and analyzed at FACS Canto II Flow Cytometer (BD Biosciences).

Blood samples were drawn from the right arm before, from the left arm after the stress test using heparinized Vacutainer® vials, and centrifuged 15 min at 1000g, 4°C, and supernatants immediately stored at -80°C. Measurements were done in duplicates using enzyme-linked immunoassay (ELISA) kits according to the manufacturers′ instructions; noradrenaline and adrenaline: Labor Diagnostika Nord, Nordhorn, Germany (detection limits 20 pg/ml noradrenaline, 5.2 pg/ml adrenaline); serotonin: DLD Diagnostika, Hamburg, Germany (5 ng/ml); oxytocin: Enzo, Lausen, Switzerland (15 pg/ml); human platelet activation factor (PAF): Abbexa, Cambridge, United Kingdom (2.5 pg/ml); human prostaglandin D2 (PGD2): Bioassay Technology, Shanghai, China (5.02 ng/ml).
Blood cells were isolated from heparinized blood samples using Ficoll-Paque Plus 1.078 g/ml density gradient (GE Healthcare Biosciences, Uppsala, Sweden). PBMCs were deposited in a Neubauer chamber and counted using Primo Vert Microscope (Carl Zeiss, Germany). PBMC subpopulations were stained using the following fluorescent labelled antibodies: CD3-APC (clone SK7; eBioscience Inc.), CD4-PE-CY7 (clone SK3; BD Biosciences), CD8-PE (clone SK1; BD Biosciences) and CD14-FITC (clone MOPC-21; BioLegend), for 30 min at 4°C; after washing, 10.000 single cell events were acquired in BD FACSCanto II with FACSDiva Software (Becton Dickinson).

Bovine beta-lactoglobulin, deferoxamine mesylate, ammonium iron (III) citrate, potassium ferrocyanide, dihidroxybenzene (catechol), dihydroxybenzoic acid (DHBA) were all purchased from Sigma (Sigma Aldrich, Steinheim, Germany). Ficoll-Paque PLUS was obtained from GE Healthcare (Uppsala, Sweden). IFNγ and IL13 ELISA kits were from ebiosciences (Santa Clara, CA). Antibodies were either purchased by ebioscience (CD3-APC clone SK7; 7AAD) or BD Bioscience (CD4-PE-Cy7, clone SK3; CD8-PE, clone SK1; Annexin V FITC).