Cxcr4 pe clone 12g5

Cells were stained using fluorescently conjugated monoclonal antibodies to CD3-PerCP (clone SP34–2), CD4-FITC (clone RPA-T4), in combination with either CCR5-PE (clone 3A9), CXCR4-PE (clone 12G5), or the activation marker CD69-PE (clone FN50), all from BD Biosciences (Franklin Lakes, New Jersey, USA). Corresponding fluorescent isotype controls were used at the same concentrations as the reference antibody. Cells were stained with antibodies by incubation for 30 min at 4°C, washed in PBS-1% FCS and fixed in 1.5% paraformaldehyde. Proliferation assays were performed using the Click-iT EdU Flow Cytometry Assay kit (Life Technologies), as per manufacturer instructions. A gate (PBMC gate) was defined in the analysis to exclude nonviable cells and debris. The percentage of live/dead cells in the PBMC gate and in the CD4⁺ cell population was analyzed using the Live/dead Fixable dead cell stain kit, as described above. Acquisition was performed with a FACScalibur flow cytometer (Becton Dickinson) and CELLQuestPro Software was used for analysis. The cell surface expression levels in the flow cytometry profiles are expressed as mean fluorescence intensity (MFI) indices. The percentage of stained cells is also presented.