Anti cd4 pe clone rpa t4

Proliferation assays used cryopreserved PBMC which were thawed, then rested overnight before labelling with Carboxyfluorescein succinimidyl ester (CFSE) as previously described [17 (link)]. Briefly, PBMC were labelled at 5–10 x 10⁶ cells/ml of pre-warmed phosphate buffered saline (PBS)/1μM CFSE at 37°C for 10 minutes, followed by quenching with five volumes of ice cold R10 and two washes. After eight days in culture with 3 μg/ml JEV peptide pools, cells were stained with near-infra-red (IR) viability dye (molecular probes), anti-CD3-AF700 (clone UCHT1), anti-CD4-PE (clone RPA-T4), anti-CD8-APC (clone RPA-T8) and anti-CD38-PE-Cy7 (clone HIT2) fluorescent antibodies (all from BD biosciences) for flow cytometry.

An aliquot of 100 μL of EDTA whole blood or 50 μL of tissue cell suspension was incubated in the presence fluorescent labeled anti-human cell surface monoclonal antibodies (anti-CD3-PerCP/CloneSK7, anti-CD4-PE/Clone RPA-T4, anti-CD8-FITC/CloneHIT8a, anti-CD25-APC/CloneM-A251, anti-FoxP3-PE/Clone259D/C7, all purchased from BD Bioscience, San Diego, CA, USA) to identify helper and cytotoxic T-cell subsets and Tregs. Following incubation, cells were treated with 1 mL of erythrocyte lysing solution for 10 min at room temperature. After one wash step with PBS, cells were fixed in MFF fix solution (10 g/L of paraformaldehyde, 10,2 g/L of sodium cacodilate and 6.63 g/L of sodium chloride, pH7.2). Stained cells were stored at 4 °C up to 24 h prior flow cytometric acquisition. A total of 10,000/100,000 events were acquired for each blood or tissue samples to quantify T-cell subsets and Tregs, respectively. A dual laser FACScalibur flow cytometer (488 nm and 633 nm) was used for data acquisition and storage as FCS files. The FlowJo software (TreeStar Inc., Ashland, OR, USA) was used for data analysis. The results were expressed as percentage of positive cells within the lymphocyte gate.

The nonspecific ADCC assay was performed as previously described, in which the mouse mastocytoma cell line P815 was used as target cells [27 (link)]. Briefly, PBMC were stimulated with P815 cells alone or P815 cells/P815-specific Abs (P815/Abs) complex (1:100 dilution of polyclonal rabbit anti-mouse lymphocyte serum, Accurate Chemical & Scientific Corp., Westbury, NY) at an E:T ratio of 10:1. Brefeldin-A (10 μg/ml, Sigma, St Louis, MO, USA), GolgiStop (5 μg/ml, BD Biosciences) and anti-CD107a PE-Cy5 (clone H4A3, BD Biosciences) were added immediately to cell medium and incubated for 6 h. Cells were fixed by 2% PFA and stained with anti-CD3 Pacific Blue (clone UCHT1), anti-CD56 PE-Cy7 (clone B159), anti-CD16 APC-Cy7 (clone 3G8), anti-CD4 PE (clone RPA-T4), anti-CD8 APC (clone RPA-T8), and anti-IFNγ FITC (clone 25,723.11). To evaluate the response time of antibody-dependent response mediated by CD56+ T and NK cells, PBMC were cocultured with P815/Abs for 2, 4 and 6 h and fixed and stained as above. All data were acquired on BD FACS Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Treestar, Ashland, OR, USA).