Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: PIR-B inhibitory activity in FcεRI-mediated Ca2+ mobilization. Indo-1/AM dye preloaded mast cells from wild-type mice (upper panel) and motheaten mice (lower panel) were analyzed by flow cytometry for intracellular Ca2+ levels in the presence or absence (data not shown) of extracellular Ca2+. Cells were stimulated with rat IgE anti-DNP (IgE), F(ab′)2 fragments of rat anti-PIR (anti-PIR), or both mAb’s (IgE + anti-PIR). F(ab′)2 fragments of rabbit anti-rat Ig antibodies were used as the cross-linking reagent.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Activity Assay, Flow Cytometry

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: PIR-B inhibitory activity in FcεRI-induced serotonin release. 3H-Serotonin preloaded mast cells from wild-type neonatal spleen (left) and motheaten mutant neonatal spleen (right) were incubated with rat IgE anti-DNP mAb plus various concentrations of F(ab′)2 fragments of rat mAb specific for PIR (filled circles) or unknown cell surface antigen (open circles). Antibody-primed cells were then triggered with F(ab′)2 fragments of rabbit anti-rat Ig antibody as a cross-linker, and the 3H-serotonin release was measured as described in Methods. The standard deviation from the mean (circle) is indicated by bars only for the points at which this value (1 SD) is greater than the circle radius.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Activity Assay, Mutagenesis, Incubation, Standard Deviation