[PubMed] [Google Scholar] 54. with a FLIPR, and data were expressed as fluorescence units versus time. Chemotaxis assay. For the chemotaxis assay, CCR5-transfected Jurkat cells (a CD4+-T-cell line with endogenous CXCR4 and stably transfected with human CCR5 [MRC, Centralised Facility for AIDS Reagents]) were preincubated for 10 min with AMD3451 at the indicated concentrations. Then 5-m-pore-size Transwell filter membranes (Costar) were loaded with 106 cells and transferred to a 24-well plate containing 100 ng of CXCL12/ml or 500 ng of CCL4/ml in 600 l of buffer. The plate was then incubated at 37C and 5% CO2 for 4 h, after which the filter inserts were carefully removed and the migrated cells were collected from the wells and fixed with 1% paraformaldehyde. Then each sample was counted for 2 min in a FACSCalibur flow cytometer, and viable cells were analyzed by the conventional forward and side scatter gating. A serial of standards (1/2 dilutions of 106 cells to 98 cells) was used to calibrate the exact amount of cells that were in the samples by linear regression. To calculate the percentage of migrated cells, the numbers of migrated cells in the compound-exposed samples were compared with the number of migrated cells in the untreated positive control (no pretreatment with AMD3451). Receptor internalization assay. U87.CD4 cells stably transfected with green fluorescent protein (GFP)-coupled CXCR4 (U87.CD4.CXCR4-GFP) were seeded in 0.001% poly-d-lysine-coated eight-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, Ill.) at 4 104 cells per well. The next day, the cells were preincubated in cell culture medium with or without 400 M AMD3451 for 15 min at room temperature. Then CXCL12 was added at a final concentration of 1 1 g/ml. After incubation at 37C for 45 min, the chamber slides were placed on ice and the cells were washed once with ice-cold PBS, fixed with 1% paraformaldehyde in PBS for 5 min on ice, and washed three Bupranolol times with ice-cold PBS. The chambers were removed from the glass slides, and a coverslip was placed on the cells. On the other hand, CEM Bupranolol cells stably transfected with GFP-coupled CCR5 were washed once with calcium flux assay buffer and preincubated with or Keratin 7 antibody without AMD3451 at 400 M for 15 min at room temperature. After 30 min of incubation at 37C with CCL3L1, added at a final concentration of 100 ng/ml, cells were placed on glass slides and Bupranolol a coverslip was fixed on the slide with nail polish. For both cell lines, cell-associated fluorescence was examined by a Nikon fluorescence microscope (Tokyo, Japan). Site-directed mutagenesis and expression of mutant receptors. Point mutations were introduced in the CXCR4 receptor by oligonucleotide-directed mutagenesis, and wild-type and mutant receptors were expressed in COS-7 cells as described previously (32, 37). The His residues, His113, His203, and His281, located in the extracellular loops or in the transmembrane domains, were individually mutated to Ala residues. In addition, four Asp residues (Asp171 [located in transmembrane domain IV TM-IV], Asp182 and Asp193 [located in extracellular loop 2], and Asp262 [located in TM-VI]) were mutated to Asn residues (32). Receptor binding assays. The human chemokine Met-CXCL12 was kindly provided by Michael A. Luther (Glaxo Wellcome). This CXCL12 contains an additional NH2-terminal methionine; however, the protein shows the same binding properties as natural ligand CXCL12 (18, 58). 125I-labeled Met-CXCL12 was prepared by oxidative iodination with IODO-GEN (Pierce), followed by high-pressure liquid chromatography purification to separate unlabeled and labeled compound. The MAb 12G5 was kindly provided by Jim Hoxie (University of Pennsylvania, Philadelphia). 12G5 was 125I-labeled by.