[PMC free article] [PubMed] [Google Scholar] 19


[PMC free article] [PubMed] [Google Scholar] 19. MO, USA) and mAb F23C49 (murine IgG2a, directed against CFTR corrector 2 [18], a gift from Dr A.H.J. Kolk) were used as isotype controls. Measurement of intracellular [Ca2+] PBMC were washed and suspended in HEPES buffer (132 mm NaCl, 6 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 12 mm K2HPO4, and 20 mm HEPES, pH 74, supplemented with 05% (wt/v) human serum albumin (CLB) and 01% (wt/v) glucose). The cells were loaded with 1M Indo-1 AM (Molecular Probes, Leiden, The Netherlands) at 37C during 1 h, washed twice, resuspended in HEPES buffer and kept on ice until use. Cells were measured at 37C on a FACStar flowcytometer (Becton Dickinson Immunocytometry Systems, Erembodegem, Belgium) equipped with an argon UV-laser and coupled to a Hewlett Packard computer equipped with LYSYS II software. Antibodies for stimulation were added as indicated. Lymphocytes were gated, based on FSC and SSC, and Indo-1 fluorescence emission at Mouse monoclonal to TYRO3 405 nm (violet) and 470 nm (blue) was recorded. The ratio of emission at 405 nm and at 470 nm was calculated and expressed as a function of time. This ratio is a measure of cytoplasmic Ca2+ concentration, which is largely independent of the cytoplasmic Indo-1 concentration. Biochemical analysis of TCR signalling PBMC were washed and suspended in HEPES buffer as described above and were stimulated with CD3 mAbs (1g/ml) at 37C. Following activation the cells were rapidly pelleted and lysed in ice-cold lysis buffer (1% Nonidet P-40; 50 mm Tris-HCl, pH 74; 150 mm NaCl; 1 mm EDTA; 1 mm PMSF; 1g/ml leupeptin; 1g/ml aprotinin; 1 mm Na3VO4; 1 mm NaF). Nuclear debris was removed by centrifugation for 15 min at 13 000 r.p.m. For immunoprecipitation, lysates were precleared with protein A-CL4B Sepharose beads (Pharmacia, Uppsala, Sweden) in the presence of nonimmune mouse or rabbit IgG. Immunoprecipitation of phosphotyrosine proteins with mAb PY-20 (Transduction Laboratories, Lexington, KY, USA) and protein A-CL4B Sepharose beads was conducted for 2 h. Immunoprecipitation of LAT with anti-LAT polyclonal rabbit antibodies (Upstate Biotechnology, Lake Placid, NY, USA) and protein A-CL4B Sepharose beads was conducted overnight. Immunoprecipitates were washed twice with ice-cold lysis buffer and eluted by boiling in reducing sample buffer. Total lysates and immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes (Hybond P, Amersham, Aylesbury, UK). After blocking (5% bovine serum albumin for CFTR corrector 2 PY-20, 5% non fat dried milk for the other Abs), phosphotyrosine proteins were detected with HRP-conjugated mAb PY-20 (Transduction Laboratories) and phosphorylated Mitogen-Activated Protein Kinases (MAPK) Erk1 and Erk2 were detected with phosphospecific p44/42 MAPK antibodies (polyclonal rabbit antibodies, New England Biolabs, Beverly, MA, USA), followed by HRP-conjugated swine antirabbit immunoglobulins antibodies (Dako, Glostrup, Denmark). Blots were developed with enhanced chemiluminescence reagent (ECL, Amersham) and autoradiography was performed. Blots of LAT immunoprecipitates were stripped after phosphotyrosine detection and probed with anti-LAT antibodies CFTR corrector 2 to confirm the identity of the precipitated phosphoprotein. Similarly, the phosphospecific MAPK blots were stripped and probed with p44/42 MAPK antibodies (polyclonal rabbit antibodies, New England Biolabs, Beverly, MA, USA). PBMC and T cell cultures PBMC or purified T cells were cultured in round bottom 96-well culture plates (Costar, Cambridge, MA, USA) at 40 000 lymphocytes in a final volume of 170l per well. The medium consisted of Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 5% heat inactivated (45min, 56C) freshly drawn autologous serum, 50M 2-ME, penicillin (100 IU/ml) and streptomycin (100g/ml). Graded amounts of CD3 mAbs, F(ab)2 fragments or isotype control antibodies were added to stimulate the cultures. As costimuli either CD28 mAb (1g/ml) or rh-IL-2 (20 IU/ml, Cetus, Emeryville, CA, USA) were added. The culture plates were incubated at 37C in a humidified atmosphere of 5% CO2. For flowcytometry, cells were harvested at timepoints as indicated. For proliferation studies, cells were cultured in triplicate for 3 days, 3H-thymidine (02Ci per well) was added for the final 4 h, and thymidine incorporation was decided on a scintillation counter. To study cytokine production, supernatants were harvested after 3 days and stored at ? 80C until further use. Monoclonal antibodies for immunofluorescence FITC-conjugated CD3 (Leu-4), CD25 and CD69, phycoerythrin (PE)-conjugated CD2, CD4 and CD27, peridinin chlorophyll protein (PerCP)-conjugated CD4 and CD8 and allophycocyanin (APC)-conjugated streptavidin, as well as appropriate isotype controls, were bought from Becton Dickinson Immunocytometry Systems. IgA-CD3 and IgG2a-CD3 can block binding of FITC-conjugated Leu-4 mAb for more than.