Antibodies as a control, and after that incubated at four . Cells were washed 3 times in PBS containing five FBS and incubated with anti-mouse IgG labeled with FITC for two h at 4 . The cells have been subsequent washed 3 times with ice-cold PBS, 5 FBS buffer, resuspended in 200 l PBS, after which analyzed by flow cytometry to figure out the surface expression levels in the receptor. Calcium flux assay Jurkat T cells had been washed twice with HBSS (Mediatech Co., Herndon, VA, USA) and resuspended at 1 106 cells/ml in HBSS. The cells had been pretreated with Slit-2 supernatant (one hundred g/ml) and BDCA-2 Proteins Purity & Documentation handle supernatant (100 g/ml) for 30 min at 37 . They had been next loaded with Indo-1 AM by adding five l working (1 g/ml/l DMSO) Indo-1 AM resolution and incubated for 45 min at 37 . The cells were then treated with CXCL12 (50 ng/ml) and analyzed for calcium mobilization by flow cytometry (FACSVantage, BD Biosciences, San Jose, CA, USA). Receptor-binding assay The binding of CXCL12 to its receptor CXCR4 was assessed by utilizing 1 ng/ml 125I-labeled CXCL12 (Amersham Biosciences, Piscataway, NJ, USA) in the presence of different concentrations of purified Slit-2 or unlabeled CXCL12 (PeproTech, Rocky Hill, NJ, USA) [29]. Briefly, Jurkat T cells at 107/ml in RPMI 1640 [containing 1 BSA (w/v) and 25 mM/ L HEPES] have been incubated inside the presence of a variety of concentrations of purified Slit-2 or unlabeled CXCL12, together with 1 ng/ml 125I-labeled CXCL12 for 1 h at room temperature and then washed three times with cold RPMI 1640 (containing 25 mM/L HEPES). Cell pelletassociated radioactivity was determined within a -counter. Preparation of PBMCs, monocytes, and CD4+ T cells Key mononuclear cells have been isolated from heparinized venous blood, as described just before [49]. Blood, collected from healthy donors, in line with a protocol, which has been approved by the Beth Israel Deaconess Medical Center Committee on Clinical Investigations, was subjected to Ficoll-Paque density gradient centrifugation at 3000 rpm for 25 min. For the main lymphocyte culture, the cells were suspended in RPMI containing 15 FCS, two mM glutamine, 50 IU/ml penicillin, and 50 g/ml streptomycin. Monocytes were depleted by two rounds of adherence to plastic. Nonadherent cells have been stimulated with phytohemagglutinin (5 g/ml) for 3 days. Cells were then removed and placed in fresh medium supplemented with recombinant human IL-2 (Advanced Biotechnologies, Columbia, MD, USA). The purity from the PBMCs was checked by flow cytometry using CD3 antibody. Two-week-old cells had been utilised for numerous experiments. For the main CD4+ T cells, PBMCs have been washed with PBS containing two BSA, and CD4+ T cells were collected by utilizing the EasyTM CD4+ T cell enrichment technique (StemCell Technologies, Vancouver, BC, Canada), based on the manufacturer’s guidelines. Briefly, CD4+ T cells were negatively isolated from a mononuclear cell sample by remedy using a CD8, CD14, CD16, CD19, CD56, TCR/, Glycophorin A, and Dextran Beta-2 Adrenergic Receptor Proteins supplier antibody mix. The antibody-coupled cells had been depleted by using magnetic Dextran iron particles. The purity was checked by flow cytometry utilizing CD4 antibody. For the primary monocytes, PBMCs had been washed with PBS containing 0.1 BSA, and after that the monocytes have been collected by using the Dynal negative-selection program (Dynal Biotech, Norway), as outlined by the manufacturer’s guidelines. Briefly, monocytes have been negatively isolated from the mononuclear cell sample by treatment using a CD2, CD7, CD16, CD19, CD56, and CD235a antibody mix.