Analysis of subclones from the pro-B line NFS70. (A) Pedigree illustrating subcloning of the cell line NFS70.255.23 from the parental NFS70 line by the limited dilution method. (B) Flow cytometric analysis of cytoplasmic Ig-μ production in NFS70 subclones. Cells were fixed, permeabilized, stained with FITC-conjugated goat antibodies against Ig-μ and analyzed by FACS. (C) Analysis of immunoprecipated and 35S-labeled Ig-μ chains from NFS70 subclones. Metabolically labeled cell lysates from the Ig-μ-negative subclone NFS70.226 and the Ig-μ-positive subclone NFS70.255 were precipitated with anti-Ig-μ (lanes μ). Precipitation with protein G sepharose only served as negative controls (lanes ø). Cell lysates were separated by 10% SDS-PAGE and radioactivity was detected in a PhosphoImager. (D) PCR-based analysis of IgH rearrangement. Schematic illustration of VH and D segments in the germline Ig locus before rearrangment and corresponding PCR primers (upper part). When VDJ rearrangement has occurred on both alleles, no 560 bp PCR amplicon can be generated. Pro-B cell line FL14.9 with one DJ-rearranged allele was used as control (lower part). (E) PCR-based analysis of JH segment usage. Schematic illustration of VH, D, and JH segments in the germline IgH locus after rearrangement and corresponding PCR primers (upper part). Depending on the JH segment combined to the D segment, different amplicon patterns can be detected on an agarose gels. JH2 is used on both alleles in subclone NFS70.255 (lower part). (F) Schematic illustration of the productively and nonproductively rearranged allele of the Ig-μ-positive subclone NFS70.255.23. Gray and colored boxes indicate protein coding sequences, white boxes untranslated exonic sequences.
