Detection of the phosphorylation of BTK, PLC2, ERK and AKT Ramos cells selleck chemicals were pretreated with RO9021 followed by stimulation with goat F 2 anti human IgM. The protein phosphorylation was detected with rabbit antibodies of anti phospho BTK, anti phospho PLC2, anti phospho ERK or anti phospho AKT. Flow cytometric analysis of CD69 upregulation in B cells Heparinized blood was collected from healthy volunteers and pre incubated with RO9021 followed by stimulation with goat F 2 anti human IgM overnight. The samples were stained with PE mouse anti human CD20 and APC mouse anti human CD69. The percen tage of activated B cells was determined using unstimulated and stimulated samples as references.

Fc receptor mediated and lipopolysaccharide mediated TNF production in human monocytes Peripheral blood mononuclear cells were isolated by centrifugation from heparinized blood in a Vacutainer CPT Cell Preparation Tube. PBMCs were cultured for 1 to 2 hours Inhibitors,Modulators,Libraries to allow monocytes to adhere, and nonadherent cells were washed away. The monocytes were stimulated with human IgG coated copo lymer microsphere beads or lipopolysaccharide for 4 hours. TNF levels in supernatants were deter mined by enzyme linked immunosorbent assay kits. IgE induced histamine release in human mast cells The method has been reported previously by our group. Briefly, human cord blood derived CD34 hematopoietic stem cells were cultured in a serum free StemPro 34 medium with stem cell factor and IL 6 for 8 weeks followed by 5 day stimulation with IL 4. For measuring histamine release, cells were sensitized with 0.

1 ug ml anti 4 hydroxy 3 nitrophenylacetyl hapten IgE overnight, and Inhibitors,Modulators,Libraries then cross linked with 1 ug ml NP BSA for 30 minutes. Supernatants were collected and assayed for histamine release using a hista mine enzyme immunoassay. The percentage of histamine release was calculated by comparing various treatments with positive control. Flow cytometric analysis of phosphorylated STAT1 and STAT5 Human PBMCs were pre incubated with compound for 30 minutes followed by 20 minutes stimulation with IL 2 for signal transducers and activators of tran scription 5 phosphorylation or IFN for STAT1 phosphorylation. Inhibitors,Modulators,Libraries For IL 2 induced STAT5 phosphorylation, cells were stained with FITC anti human CD3 and Alexa Fluor 647 anti STAT5, and quanti tated pSTAT5 fluorescence intensity gated on the CD3 T cell population.

For IFN induced STAT1 phospho rylation, cells were stained with PE anti human CD14 and Alexa Fluor Inhibitors,Modulators,Libraries 647 anti STAT1, and quantitated pSTAT1 fluorescence intensity gated on CD14 monocytes macrophages. Mouse Inhibitors,Modulators,Libraries bone marrow macrophage derived osteoclastogenesis Bone marrow cells were obtained selleck chem from C57BL 6 mouse tibiae and suspended in culture medium supplemented with monocyte colony stimulating factor for 16 hours.