For FACS sorting, spleens pooled from either immune (m+s)Ig mice (2 mice per sort, 2 sorts per time point) or immune non-Tg B6 mice (2 mice per sort, 3 sorts 13–15 wk after immunization) were treated with unlabeled anti-FcγRII/III (24.G2) and stained with Alexa Fluor 488–labeled B220 (RA3-6B2), PE-labeled CD80, and NIP-haptenated allophycocyanin (NIP-APC). Alternatively, cells were stained with FITC-labeled anti-CD35 (8C12; BD Biosciences), anti–CD80-PE, NIP-APC, and APC/Cy7-labeled anti-B220 (RA3-6B2; eBiosciences). Propidium iodide was used for live/dead discrimination. Cells were sorted on either a FACSVantage or FACSAria (BD Biosciences). Cell pellets were digested overnight at 37°C in 10 μl of digestion buffer (50 mM Tris, pH 8.0, 50 mM KCL, 0.63 mM EDTA, 0.22% NP-40, and 0.22% Tween-20) containing 0.8 μg/ml proteinase K (Novagen). Vλ1 sequences were amplified by nested PCR using Pfu Turbo polymerase (Stratagene) using external primers 5′-GCACCTCAAGTCTTGGAGAG-3′ and 5′-ACTCTCTCTCCTGGCTCTCA-3′ and internal primers 5′-CTACACTGCAGTGGGTATGCAACAATGCG-3′ and 5′-GTTCTCTAGACCTAGGACAGTCAGTTTGG-3′. Amplified DNA was cloned directly into pCR 4 Blunt-TOPO vector using the Zero Blunt TOPO PCR Cloning kit for sequencing (Invitrogen). Vλ1 DNA was further amplified by placing colonies directly into PCR reactions containing the primers M13 forward 5′-GTAAAACGACGGCCAG-3′ and M13 reverse 5′-CAGGAAACAGCTATGAC-3′. DNA was purified from the PCR reaction mixture with the QIAquick PCR Purification kit (QIAGEN), mixed with sequencing primer, T3 5′-AATTAACCCTCACTAAAGGG-3′, and sequenced by the Keck Biotechnology Resource Laboratory at Yale University School of Medicine using Applied Biosystems DNA sequencers. Sequences were aligned to a rearranged germline Vλ1/Jλ1 sequence using Lasergene DNA analysis software.