Membrane components were prepared at 5 107 cells/ml using 1% NP-40 in PBS with 1 mM AEBSF, 1 M pepstatin A, 10 M E-64, 10 M bestatin, 100 M leupeptin, and 2 g/ml aprotinin (lysis buffer). binding and the presence of CD32 were recognized by mAb and analyzed by GR 103691 two-color circulation cytometry. Cells expressing CD32 bound CRP inside a dose-dependent and saturable manner consistent with receptor binding. CRP bound to transfectants and K-562 cells with related kinetics, and in both instances binding was completely inhibited by aggregated IgG. On monocytic cell lines, treatment with Bt2cAMP improved FcRII manifestation and enhanced CRP binding. CRP also specifically precipitated FcRI and FcRII from your monocytic cell collection, THP-1. It is suggested that the major receptor for CRP on phagocytic cells is definitely FcRII. for 10 min at 21C and then incubated at 37C for 5 h. Cells were cultured over night in DMEM with 10% FCS. 18 h after transfection, cells were detached using PBS comprising 5 mM EDTA and replated on 100-mm cells tradition plates (two 100-mm cells tradition plates for cells from each 6-well plate) and cultured in DMEM. Circulation cytometric assays were performed 66C78 h after transfection. The percentage of transfected cells was 75C85% as identified using either FITC-FLI8.26 or PE-C1KM5. CRP Binding Assay. Cells were washed twice in ice-cold PBS comprising 0.05% azide and 0.1% globulin-free BSA (PAB). Between 5 105 and 106 cells per tube were washed with 1 ml of ice-cold PAB. Cells were pelleted at 180 for 5 min at 4C and resuspended in PAB with CRP in the concentrations indicated in a total volume of 80 l. Cells were incubated for 1 h in the presence of CRP, then washed twice with 1 ml of PAB. Cells were then incubated for 30 min at 4C with mAb 2C10 tradition supernatant. Cells were washed twice with 1 ml PAB and then incubated with PE-GAM (1 g/106 cells) or FITC-GAM (a 1:100 final dilution) for 30 min. After this last incubation, cells were washed twice with 1 ml PAB as above and then resuspended in 0.4C0.5 ml PAB for analysis by flow cytometry. FcRI levels were identified using mAb 32.2 and PE-GAM. Levels of FcRII were determined by binding of FITC-AT10. The percentage of deceased cells was identified using 7-Aminoactinomycin D (Molecular GR 103691 Probes). To test inhibition by aggIgG, cells were incubated with aggIgG at an increasing concentration for 30 min before the addition of CRP. There was no significant switch in the fluorescence of the secondary antibodies in the presence of GR 103691 aggIgG. Circulation Cytometry. Cells were analyzed using a Becton Dickinson FACSCalibur? circulation cytometer equipped with CellQuest software (Becton Dickinson). The population analyzed was gated by ahead and part scatter to exclude deceased cells. A minimum of 30,000 cells was collected. For those measurements of CRP binding, the binding of 2C10 and the antiCmouse secondary antibody were subtracted. Results are offered as the geometric mean channel fluorescence (gMCF). Precipitation of Cell Surface Receptors. THP-1 cells were treated with 50 g/ml pronase (Calbiochem) for 30 min at 37C, washed, and radiolabeled with 125I Fam162a using lactoperoxidase 14. Membrane components were prepared at 5 107 cells/ml using 1% NP-40 in PBS with 1 mM AEBSF, 1 M pepstatin A, 10 M E-64, 10 M bestatin, 100 M leupeptin, and 2 g/ml aprotinin (lysis buffer). CRP beads were prepared by coupling 18 mg CRP/ml Affigel 15 according to the manufacturer’s protocol. Antibody beads were prepared by incubation of 5 g anti-CD32 mAb (IV.3 or AT10) or anti-CD64 mAb 197 with 15 l protein ACSepharose. 2.5 106 cell equivalents were incubated with CRP beads, anti-CD32 Sepharose, or anti-CD64 Sepharose in lysis buffer for 4 h at 4C. The beads were then washed four instances with lysis buffer and once with 0.1% NP-40 in the same buffer. The samples were boiled for 3 min in Laemmli’s sample buffer and run on 4C20% gradient SDS-PAGE gels (Novex). The gels were dried and autoradiography was performed using a Storm imaging system (Molecular Dynamics). Data Analysis. The binding kinetics of CRP to cells were analyzed using GraphPad PRISM? software (GraphPad Software, Inc.). ImageQuant software (Molecular Dynamics) was utilized for quantitation of radioactive bands. Experiments were repeated at least twice. Results Binding of CRP to Transfected COS-7 Cells and to K-562 Cells. Transfection of COS-7 cells with pcDSR296 comprising the cDNA for CD32 resulted in 70C85% of cells expressing the CD32 marker. When these cells were incubated with CRP, a dose-dependent and saturable binding of CRP was seen (Fig. 1 A). The binding of CRP was fitted to a single site model using.