Apc mouse anti human cd4 antibody

HEK293T (ATCC®-Number CRL-11268), the HIV reporter cell line LC5-RIC, the human T-lymphoma cell line H9 (ATCC®-Number HTB-176) and peripheral blood mononuclear cells (PBMC) were cultured as described in^{6 (link),8 (link)}. PBMCs were isolated from buffy-coats (received from the Blood Donation Services of the Bavarian Red Cross GmbH) by Ficoll density gradient centrifugation as described in^{8 (link),45} . Prior to infection, PBMCs were stimulated with 20 U/ml hIL-2 and 1 µg/ml phytohemagglutinin (PHA) for 72 h. CD4+ cells were enriched from whole blood using the RosetteSep^TM Human CD4+ T Cell Enrichment Cocktail from Stemcell Technologies according to the manufacturer's instructions and cells handled like PBMCs. CD4+ cell enrichment was verified by staining with APC mouse anti-human CD4 antibody (BD Pharmingen) and FACS analysis, yielding ~94% CD4+−positive cells.
All cells were cultured under standard conditions, 37 °C and 5% CO₂.

To study the phenotype of H1.ME-DCs, the cells were stained with antibodies against CD11c, CD40, CD83, CD86, HLA-DR and HLA-A2 (BD Biosciences) and analyzed with a FACSCalibur flow cytometer (BD Biosciences). To check the phenotype of MART-1-specific CD8+ T cells after multiple stimulations, the cells were stained with R-PE-labelled A*0201/ELAGIGILTV Pentamer and antibodies against CD8, CD45RA and CD62L (BD Biosciences) before analysis using FACSAria flow cytometer.
To measure the allostimulatory function of DCs, frozen human peripheral blood pan-T cells were thawed and labelled with Carboxyfluorescein diacetate succinimidyl ester (CFSE; Life Technologies) as described previously9 (link). To set up allostimulation assay, 2 × 10⁵ CFSE-labelled pan-T cells were co-cultured with DCs at various DC:T cell ratios. After 5-day incubation, the samples were stained with APC mouse anti-human CD4 antibody (BD Biosciences) and the CD4+ T cell proliferation was evaluated by CFSE dilution after gating on CD4+ population using FACSAria flow cytometer.