Fitc conjugated anti igg1

Lymphocytes of the thymus, bone marrow, spleen, and LN from 4–6-wk-old mice of the described genotypes were made into ∼1 × 10⁵ cells ml⁻¹ single-cell suspensions, which were stained using fluorescence-conjugated antibodies before analyzed by flow cytometry. For the CSR assay, CD43⁻ splenic B cells were purified using anti-murine CD43 magnetic beads (MACS; Miltenyi Biotech) and cultured at 0.5 × 10⁶ cells ml⁻¹ in RPMI medium supplemented with 15% FBS and 25 ng ml⁻¹ of LPS plus 25 ng ml⁻¹ of IL-4 (R&D). Cultured cells were maintained daily at a density of 0.5–1.0 × 10⁶ cells ml⁻¹. Cells were collected 2–4 d after activation for flow cytometry with FITC-conjugated anti-IgG1 (BD PharMingen) and Cyc3-conjugated anti-murine B220 (eBioscience). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Bioscience) and the data processed using FlowJo software package.