phosphatase substrate buffer consisting of 1 mg/ml 4­Nitrophenyl phosphate disodium salt hexahydrate (Alkaline Phosphatase Substrate; Sigma­Aldrich) diluted in 1 M diethanolamine, 8.4 mM MgCl2, and 0.02% NaN3, pH 9.8, and absorbance values read at 405 nm (VersaMax ELISA reader; MDS Analytical Technologies). The following NP­ specific mouse mAbs were used as standards to quantify the absolute concentration of NP­specific anti ­ body present in the sera of immunized mice: B1­ 8  (NP­ specific IgM; Reth et al., 1978), S24/63/63 (NP­specific IgG3; Baumhäckel et al., 1982), N1G9 (NP­ specific IgG1; Cumano and Rajewsky, 1985), S43­ 10 (NP­ specific IgG2c; Reth et al., 1978), and D3­ 13F1 (NP­ specific IgG2b; gift from K. Rajewsky). In the cases where a standard was unavailable, the titer at an OD50 was determined from the serial dilution curves for each sample. For both adoptive transfer and mixed bone marrow chimera analyses, detection of NP­ specific IgG2a or IgG2c serum antibodies by ELISA was accomplished with AP­ conjugated AffiniPure goat anti–mouse IgG, Fc Subclass 2a–specific (Jackson ImmunoResearch Laboratories), or AP­ conjugated AffiniPure goat anti–mouse IgG, Fc Subclass 2c–specific (Jackson ImmunoResearch) antibodies, respectively. The detection of NP­ specific IgMa or IgMb serum antibody responses was accomplished as previously described (Rubtsov et al., 2005) with either biotin­ conjugated mouse anti­ IgMa (DS­ 1; BD) or biotin­ conjugated mouse anti­ IgMb (AF6­ 78; BD) antibodies, respectively. Plates were subsequently washed three times and bound antibody revealed by incubation with AP­ conjugated Streptavidin (Southern Biotech). For affinity maturation measurements, ELISAs were performed as in the previous paragraphs with the exception that plates were coated with either NP (4­Hydroxy­3­nitrophenylacetyl) 4–BSA or NP16–BSA capture antigens (Biosearch Technologies) diluted at 2 µg/ml. The ratio of high­affinity anti ­ bodies bound to NP4–BSA relative to the total of both high­ and low­ affinity antibodies bound to NP16–BSA was calculated as previously described (Herzenberg et al., 1980; Cumano and Rajewsky, 1985). In brief, the rela­ tive NP4/NP16 binding ratio for a given sample was calculated as the OD50 titer of serum antibodies bound to NP4–BSA divided by the OD50 titer of antibodies bound to NP16–BSA. The NP­specific mAb S43­10 (Reth et al., 1978) was used as a positive control for a high­affinity NP­specific IgG2c antibody with an affinities of Kd = 2.4 × 108 M for NIP and Kd = 2.8 × 107 M for NP (Cumano and Rajewsky, 1985). NP-specific ELISPOTS. NP­specific ASCs were measured in 96­well flat­bottom EIA/RIA high­binding plates (Costar; Corning) coated over ­ night at 4°C with 2 µg/ml NIP15­BSA diluted in 0.05 M K 2HPO4, pH 8.0. Plates were washed three times with PBS before blocking with warm PBS and 1% gelatin (Sigma­Aldrich) at 37°C for a minimum of 1 h. Plates were washed again three times with PBS before incubation with cells. Cells were isolated from spleen, bone marrow, or lymph nodes at the times indicated after immunization and seeded in duplicate at a starting density of 0.25–1 × 107 total viable cells per 100 µl in the first well, and threefold serial dilutions were performed down the plate. For those ELISPOTs performed on lymph node cells, lymph nodes harvested from three mice within the same treat­ ment group were pooled together to constitute a single sample. Cultured cells were incubated at 37°C in 5% CO2 for 6 h in complete IMDM (Invit­ rogen) supplemented with 10% heat­inactivated FBS (Biosource, Invitro ­ gen, or Gemini Bio­Products), 2 mM GlutaMAX­I (Invitrogen), 100 U/ml Penicillin (Invitrogen), 100 µg/ml Streptomycin (Invitrogen), and 0.05 mM 2­mercaptoethanol (Sigma­Aldrich). Plates were then washed once with H2O and 0.05% Tween 20 for 10 min at room temperature and subse­ quently three times with PBS and 0.1% Tween 20. Secreted antibody was detected by incubating plates with an isotype­specific AP­conjugated goat anti–mouse Ig (SouthernBiotech) diluted in warm PBS, 1% gelatin, and 0.05% Tween 20 for 1 h at 37°C. After three washes (PBS, 0.1% Tween 20), plates were developed overnight at 4°C with 1 mg/ml 5­ Bromo­ 4­ chloro­3­indolyl phosphate p­toluidine (Sigma­Aldrich) salt substrate diluted in an alkaline buffer composed of 0.1 M 2­amino­2­methyl­1­propanol, 0.01% NaN3, 0.5 mM MgCl2, and 0.007% Triton X­ 405, pH 10.25. Plates were washed four times with deionized H2O, allowed to dry in the dark at room temperature, and scanned (Perfection 2450 Photo Scanner; 1496 Epson). Developed spots were counted visually from the scanned images and the frequency of NP­ specific ASCs per total number of cells plated was enumerated. Splenectomy operations. For both vital splenectomy and sham opera­ tions, 6­ wk­ old C57BL/6 mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine, followed by administration of a subcutaneous injection of 0.5 mg/kg buprenorphine for pain. Once sedated, a 1–1.5­cm incision was made on the upper left abdomen through both the peritoneal wall and skin. For splenectomized animals, the spleen was exteriorized and the splenic artery and efferent venule were each ligated. For sham­operated mice, the spleen was gently replaced back into the abdo ­ men. For all animals, the abdominal wall was closed by placing one to two sutures through the peritoneal membrane, and subsequently two wound clips were placed to adjoin the exterior skin of the incision. Postoperative care included administration of 1 ml of subcutaneous saline, rest near an in­ direct heat source, and monitoring until full motility was regained. Pain management was achieved by subcutaneous injections of 0.5 mg/kg bu­ prenorphine every 12 h for 2–3 d after surgery. Mice were allowed to re­ cover for 21–29 d after surgery, at which time the total serum IgM was determined to be equivalent between the sham­operated and splenecto ­ mized groups. At 23–32 d after operation, mice were immunized and serum NP­specific antibody responses measured by ELISA as described. MZ B cell depletion. For selective MZ B cell depletion in vivo, a protocol was used that was similar to a previously published protocol (Belperron et al., 2005, 2007). In brief, C57BL/6 mice were injected i.p. with 100 µg each of unconjugated purified rat IgG2a anti–mouse CD11a (Integrin L, LFA­1 ; clone M17/4; hybridoma) and rat IgG2b anti–mouse CD49d (Integrin  ; 4 VLA­4; clone PS/2; hybridoma) mAbs diluted together in sterile PBS. Con ­ trol mice were injected with 100 µg each of functional grade purified rat IgG2a (clone eBR2a; eBioscience) and rat IgG2b isotype control antibodies (clone eB149/10H5; eBioscience) also diluted in PBS. 3 h after injection of antibodies, mice were bled and PBLs stained for the mature B cell markers CD21 and CD1d to verify MZ B cell release into blood. Greater than 80% of MZ B cells are depleted entirely from the spleen up to 10 d after injection of anti­integrin antibodies (Fig. S2 A), and by 14 d after treatment >60% of normal frequencies of MZ B cells have returned to spleen (Fig. S2 A). Approximately 10% of the anti­integrin antibodies injected remain within circulation 4 d after injection and are essentially completely cleared by 7 d after injection (Fig. S2 C). However, surface integrin expression is also down­ modulated by the anti­ 4/L treatment on remaining T and B lymphocytes up to 4 d after injection (Fig. S2D). Thus, for MZ B cell depletion experi­ ments, mice were rested for 7 d after injection of anti­integrin antibodies and before immunization (Fig. S2 E) to allow both for clearance of rat anti­ 4/L antibodies (Fig. S2 C) and for remaining T and B lymphocytes to regain wild­type levels of surface integrin expression (Fig. S2 D). 7 d after injection of antibodies, mice were immunized with NP­Ficoll ± poly(I:C) as described in Fig. S2 E. Sera were collected either the day before immuni­ zation (preimmune) or at 4 and 7 d after immunization, and serum NP­ specific antibody responses were measured as described. At 7 d after immunization, splenocytes were harvested and the frequency of NP­specific ASCs was evaluated by ELISPOT. Depleting anti­integrin antibodies were tested by the LAL assay and found to be at levels ≤0.1 EU/ml. To determine the clearance of serum rat IgG antibodies over time in this model, serum was collected from mice injected with either rat IgG anti­ integrin and isotype control antibodies and analyzed for the presence of rat IgG by ELISA. In brief, plates were coated with 10 µg/ml of unlabeled goat anti–rat IgM + IgG (H + L chain specific, absorbed against mouse Ig) anti­ body in PBS overnight at 4°C. After washing (PBS and 0.1% Tween 20), plates were blocked for 3 h at 37°C with preimmune C57BL/6 mouse serum diluted 1:200 in PBS, 1% BSA, and 0.05% NaN3 and subsequently washed twice. Sera and standards were diluted similarly as above and incubated over­ night at 4°C. Plates were washed three times, and bound antibodies were detected by incubation with AP­ conjugated goat anti–rat IgG (H + L chain IFN- drives FO B cell TI-2 responses | Swanson et al.