Anti hcd4 pe

Peripheral blood samples were collected into ethylenediaminetetraacetic acid-treated tubes by venipuncture from the subjects after an 8-h fasting and were layered on the Ficoll-Paque Plus solution (Amersham Biosciences, Amersham, Bucks, UK) in a centrifuge tube, centrifuged at 400 × g for 20 min at 21°C, and peripheral blood mononuclear cells (PBMCs) were harvested. Then, divalent cation-free Hanks balanced salt solution was used for washing of cells at 300 × g for 5 min at 4°C. PBMCs were resuspended at 10⁶ cells/ml in RPMI-1640 medium and prepared for the following procedures.
Freshly processed human PBMCs were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin in the presence of 5 μg/ml brefeldin A for 5 h at 37°C as described by others.[29 (link)] The cells were harvested and stained with anti-hCD4-PE (BD Biosciences, San Jose, California, USA) and anti-hCD8-Percp (BD Biosciences) for 30 min at room temperature, followed by staining with anti-hIL-17A-FITC (eBioscience, San Diego, California, USA) and anti-hIFN-γ-APC (eBioscience) after fixation and permeabilization. CD8⁺ subpopulations were determined using FACS-Calibur (BD Biosciences). A total of 1 × 10⁵ events were collected for each subject and data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).