Adoptive transfers were performed by intravenous injection of 5 × 103 Igλ-enriched B1-8 B cells into the retro-orbital plexus of anesthetized mice. Mice were immunized the following day. All immunizations were performed using NP-CGG (Biosearch Technologies) resuspended at 1 mg/ml in D-PBS and mixed 50:50 volumetrically with Alhydrogel (Accurate Chemical and Scientific). Mice were injected subcutaneously with 20 μl of this solution (10 μg of NP-CGG per injection) in each ear. At endpoint, the facial LNs from each side were pooled for analysis (see figure legends for various timepoints).
Unmodified αIgE (clone R1E4; produced by hybridoma culture as described below), αIgE with a mutated Fc-receptor binding domain (clone R1E4; Cedarlane), or control rat γ globulin were diluted in D-PBS to a concentration of 0.3 mg/ml and injected intravenously to achieve a final dose of 3.25 mg/kg. For the experiment shown, mouse γ globulin (Jackson ImmunoResearch) was used as a control whereas in previous experiments that the presented data are representative of rat γ globulin was used as a control.
Tamoxifen was dissolved at 50 mg/ml in corn oil (Sigma-Aldrich) by shaking at 56°C for several hours. Approximately 100 μl/mouse was delivered by intraperitoneal injection to achieve a dose of 200 mg/kg.
Unmodified αIgE (clone R1E4; produced by hybridoma culture as described below), αIgE with a mutated Fc-receptor binding domain (clone R1E4; Cedarlane), or control rat γ globulin were diluted in D-PBS to a concentration of 0.3 mg/ml and injected intravenously to achieve a final dose of 3.25 mg/kg. For the experiment shown, mouse γ globulin (Jackson ImmunoResearch) was used as a control whereas in previous experiments that the presented data are representative of rat γ globulin was used as a control.
Tamoxifen was dissolved at 50 mg/ml in corn oil (Sigma-Aldrich) by shaking at 56°C for several hours. Approximately 100 μl/mouse was delivered by intraperitoneal injection to achieve a dose of 200 mg/kg.
