Anti ccr4 pe

We first isolated peripheral blood mononuclear cells (PBMCs) from peripheral venous blood by density gradient centrifugation. PBMCs were incubated in a modified RPMI medium (calcium concentration set to 2 mM) with conjugated anti-human monoclonal antibodies in order to differentiate T-lymphocyte subsets. We applied the following anti-human monoclonal antibodies: anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CXCR3 APC, and anti- CCR4 PE (all from BD Biosciences, San Diego, CA, USA). A portion of PBMCs was set apart for measurement of Kv1.3 channel expression, and these samples were additionally incubated with anti-Kv1.3 FITC (Sigma-Aldrich, St. Louis, MO, USA). The remainder of PBMCs were loaded with the calcium-sensitive fluorescent dyes Fluo-3 and Fura-Red (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. We then divided the PBMCs into 3 equal aliquots. The first aliquot was used as a control. In the second aliquot, Kv1.3 channels were blocked with margatoxin (MGTX, 60 nM), while in the third aliquot, IKCa1 channels were blocked with triarylmethane (TRAM, 60 nM). Samples were run on a Partec CyFlow ML flow cytometer (Munster, Germany). PBMCs were activated by the addition of 20 µg phytohemagglutinin (PHA), after the acquisition of a baseline for 2 min. Kinetic cell fluorescence data were recorded for another 10 min following activation.

Measurements were taken as described earlier [3] . Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by a standard density gradient centrifugation from 9 mL of freshly drawn peripheral venous blood and afterward kept in RPMI medium supplemented with CaCl2 (calcium concentration: 2 mM) throughout the following steps of the procedure. PBMCs were then incubated with the following conjugated anti-human monoclonal antibodies: anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CXCR3 APC (for the determination of Th1 cells) and anti-CCR4 PE (for the determination of Th2 cells) (all from PharMingen, San Diego, CA, USA) according to the manufacturer's instructions. For monitoring cytoplasmic calcium levels, PBMCs were loaded with calcium-sensitive Fluo-3 and Fura Red dyes according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA, USA). PBMCs were incubated in the presence or absence of 4 nM MGTX or 240 nM TRAM. After recording a baseline for 2 min, cells were activated with 20 pg phytohemagglutinin (PHA) and the measurement was continued directly afterward in a kinetic manner for 15 min employing a flow cytometer (Beckman Coulter Gallios™, Miami, FL, USA). Recordings were evaluated with specific software (FacsKin, Budapest, Hungary) which is based on the calculation of a logistic function for each measurement [4] .