Cd3 apc clone sk7

To peripheral blood mononuclear cells (PBMCs) (0.5 Mio/test), a final concentration of 0.75 ng/ml phorbol 12-myristate 13-acetate (PMA), 100 μg/ml apo-Bet v 1 in the presence or absence of 30 μm catechol (Sigma) and 10 μm iron was added. Controls included PMA alone or in the presence of 30 μm catechol (Sigma) and 10 μm iron. PMA concentration was determined in pre-experiments and considered optimal when cells were slightly down-regulating surface CD4⁺ expression (27 (link)). After 18 h supernatants were collected and stored at −80 °C until further analysis.
Cells were stained for 30 min at 4 °C with CD3-APC (clone SK7, eBioscience (Santa Clara, CA)), CD4-PE-Cy7 (clone SK3, BD Biosciences), and CD8-PE (clone SK1, BD Biosciences), in PBS containing 2% FCS followed by a 10-min incubation of annexin V FITC (BD Bioscience) and 7-amino-actinomycin D (eBioscience) in binding buffer (10 mm Hepes, 140 mm NaCl, 2.5 mm CaCl₂) at room temperature. Acquisition and analysis were performed on a FACSCanto II machine (BD Biosciences) using the FACSDiva Software 6.0.

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).