Streptavidin rpe

The monoclonal antibodies used were purchased from eBioscience (San Diego, CA, USA) BioLegend (London, UK) or BD (San Diego, CA, USA). Flow cytometric analysis or cell sorting was performed on an LSR-Fortessa or a FACS-Aria-III, respectively (both BD). Antibodies used for flow cytometry: T-cells: 53-6.7, anti-CD8 (25-0081-82, eBioscience); RM 4-5, anti-CD4 (45-0042-82, eBioscience); 3C7, anti-CD25 (12-0251-83, eBioscience); B-cells: RA3-6B2, anti-B220 (103224, Biolegend); 1B11, anti-CD43 (121204, Biolegend); 2B8, anti–c-Kit (105813, Biolegend); II/41, anti-IgM (406509, Biolegend); 11/26C, anti-IgD (12-5993-83, eBioscience); 6D5, anti-CD19 (115533, Biolegend); myeloid cells: RB6-8C5, anti–Gr-1 (108404, Biolegend); S7, MI/70, anti–Mac-1 (17-0112-83, eBioscience). Biotinylated antibodies were detected using streptavidin-RPE (DAKO, Glostrup, Denmark) or streptavidin coupled to PE-Cy7, APC, APC-Cy7 or PerCP5.5 (BioLegend or eBioscience).

Tumors were analyzed by flow cytometry using the following cell surface markers for (a) B cells: RA3-6B2, anti-B220; R2/60, anti-CD43; II/41, anti-IgM; 11/26C, anti-IgD; MB19-1, anti-CD19; 53-7.3, anti-CD5 and B3B4, anti-CD23; 7E9, anti-CD21 (BioLegend, Fell, Germany); and (b) T cells: GK1.5, anti-CD4; H57-597, anti-TCRb (all from eBioscience, Vienna, Austria) and 53-6.7, anti-CD8; (from BD Pharmingen, San Diego, CA, USA). Biotinylated antibodies were monitored with streptavidin-RPE (DAKO, Vienna, Austria) or streptavidin-PE-Cy7 (BD Phamingen). Using a FACS^Vantage cell sorter (Becton Dickinson, Heidelberg, Germany) premalign pre/pro-B cells (B220⁺/IgM⁻) or immature (IgM⁺/IgD^low) and mature (IgM⁺/IgD^high) B cells were isolated and sorted from bone marrow and spleen, respectively. Flow cytometry data were analyzed using Cyflogic free ware and FlowJo (Ashland, OR, USA).