Journal: The Journal of Experimental Medicine

Article Title: Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

doi: 10.1084/jem.20031523

Figure Lengend Snippet: μH chain is detectable on the surface of λ5 −/− , VpreB1 −/− VpreB2 −/− , and SLC −/− pre–B cells. (A) CD19 + BM cells were isolated by MACS from tet-μHC, tet-μHC λ5 −/− , and tet-μHC VpreB1 −/− VpreB2 −/− mice that had received Tet in drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19 + BM cells were isolated by MACS from SLC −/− or Rag1 −/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.

Article Snippet: For intracellular stainings, cells were fixed and permeabilized using the FIX & PERM ® kit (Caltag Laboratories) and the following antisera: FITC- or Cy™5-conjugated polyclonal goat anti–mouse IgM (μH chain specific) and FITC- or Cy™5-conjugated polyclonal donkey anti–rabbit IgG (Dianova); FITC-conjugated anti–κL-chain and anti–λ 1 +λ 2 L-chain mAbs (BD Biosciences); polyclonal rabbit anti-TdT (Supertechs); and polyclonal rabbit anti-Ku70 (DPC Biermann).

Techniques: Isolation, Staining, Amplification, Flow Cytometry, Labeling