specific, absorbed against mouse Ig; SouthernBiotech) diluted in PBS, 1% BSA, and 0.05% NaN3 buffer for 1 h at 37°C. After three washes, plates were developed and absorbances read according to that described for the NP­ specific ELISA protocol. Purified unlabeled rat IgG antibody (Southern­ Biotech) was used as a standard to quantify the concentration of total serum rat IgG for these ELISAs. Adoptive cell transfers. Lymph node cells were harvested and purified from BL6­Igh B1­8/B1­8 ;Igha mice in IMDM supplemented with 5% FBS, washed twice (PBS), and resuspended in sterile PBS. Approximately 5 × 107 total lymph node cells were injected intravenously into each sex­matched C57BL/6­Igh b recipient mouse. 1 d after cell transfer, recipient mice were immunized with NP­Ficoll ± poly(I:C) as described. Serum was collected before adoptive cell transfer (preimmune) and at 4 and 7 d after immuniza­ tion, and NP­specific antibody responses were subsequently measured by ELISA. For phenotypic confirmation, mice were left unimmunized and ver­ ification of transferred IgMa ­expressing B cells within the splenic FO B cell compartment of C57BL/6­Igh b recipient mice 1 d after transfer was vali­ dated by flow cytometry. Mixed bone marrow chimeras. Bone marrow cells were isolated from 5–7­wk­old C57BL/6 (Igh b), B6.C20 (Igha), Tlr3/ (Ighb), or Ifnar1/ (Ighb) donor mice in IMDM supplemented with 5% FBS under sterile con­ ditions. Donor bone marrow cells were washed once with sterile PBS (Cell­ gro; Mediatech, Inc.), resuspended at 1.33–2.67 × 107 cells/ml in PBS, and mixed (Ighb + Igha) to yield a final concentration of 2 × 107 cells/ml for re­ constitution. A total of 4 × 106 bone marrow cells were injected intrave­ nously into each sex­matched lethally irradiated (1,000 rad) 6–8­wk­old B6.C20 recipients. Recipient mice were maintained on Septra food for 7–8 wk after reconstitution. At 8 wk after reconstitution, PBLs from chimeric mice were analyzed by flow cytometric analysis to ensure no significant differ­ ences were observed between the ratio of IgMb:IgMa B cell reconstitution frequencies in the experimental chimeras as compared with the control chi­ meras. Chimeric mice were immunized at 9 wk after reconstitution and serum NP­specific antibody responses were measured by ELISA as described. At 14 d after immunization, the ratio of IgMb:IgMa B cell reconstitution frequencies was determined within the chimeric splenic mature B cell com­ partments and found to be identical to those previously measured in the circulating B cell population. In vitro stimulation. B cells were negatively enriched from naive C57BL/6 mice by depletion of CD43+ cells with magnetic cell sorting on LS columns (Miltenyi Biotec) with MACS buffer (PBS, 2 mM EDTA, and 0.5% BSA) according to the manufacturer’s protocol. MZ (CD21high CD1dhigh) and FO (CD21int CD1dint) B cells were subsequently sorted based on surface expres­ sion of CD21 and CD1d to ≥94% purity using a MoFlo XDP Cell Sorter (Beckman Coulter) with Summit Software version 5.1.3.6886. Sorted MZ and FO B cells were cultured separately at 2.5–5 × 105 cells/ml in 1 ml of com­ plete IMDM in 24­well flat bottom cell culture plates (CELLSTAR; Greiner Bio­One) with the indicated stimuli at 37°C in 5% CO 2. Stimuli and their concentrations cultured with cells included the following: 1–100 U/ml re­ combinant mouse IFN­ 11 (PBL InterferonSource), 1–10 µg/ml AffiniPure F(ab)2 goat anti­mouse IgM,  chain specific (anti­IgM; Jackson Immuno­ Research Laboratories), and 0.1–10 µg/ml LPS (Escherichia coli O26:B6; Sigma­ Aldrich). B cell activation was subsequently assessed as CD69 and CD86 cell surface marker up­ regulation measured by flow cytometry after stimulation for 6 or 10 h, respectively. Endotoxin levels of stimuli used for in vitro cultures were either measured by the LAL assay or purchased as <1 EU/µg. Immunofluorescence histology. Spleens were harvested from mice 7 d after immunization and frozen in OCT compound (EM Sciences) at 80°C. Tissues were cut into 5–7­µm sections and allowed to dry at room tempera ­ ture. Unfixed sections were rehydrated with PBS for 20 min and blocked for 15–30 min with rat anti–mouse FcR blocking antibody (2.4G2) diluted JEM VOL. 207, July 5, 2010 A r t i c l e in PBS, 2% BSA, and 0.05% Tween 20 at room temperature. Sections were stained with antibody mixtures diluted in PBS for 30–60 min at room tem­ perature in the dark, followed by three 5­min washes with shaking with PBS. Incubation and washing steps were repeated in the cases where a sec­ ondary detection reagent was used. Sections were dried, mounted with Bion IFA mounting medium (Bion Enterprises, Ltd.), visualized at room tempera­ ture with a microscope (Axiovert 200M; Carl Zeiss, Inc.) using a 3i Marianis System, and analyzed with Slidebook 4.0 software (Intelligent Imaging In­ novations). Antibodies and secondary reagents used for immunofluorescence histological detection included the following: FITC­conjugated rat anti– mouse CD169 (MOMA­1; AbD Serotec), Alexa Fluor 546–conjugated rat anti–mouse IgM (R33­24.12; our laboratory), Alexa Fluor 647–conjugated mouse anti–mouse IgD (1.3–5; our laboratory), biotin­conjugated rat anti– mouse Lambda (JC5­ 1; SouthernBiotech), streptavidin–FITC (BD), and Cy5­ conjugated goat anti–mouse IgG, Fc Subclass 2c specific (Jackson ImmunoResearch Laboratories). All Alexa Fluor–conjugated antibodies were purified and conjugated in our laboratory using Alexa Fluor Protein Labeling kits (Invitrogen). Statistical and data analyses. Data were graphed and analyzed using Prism version 5.0a (GraphPad Software). Statistical significance was assessed with a Student’s t test with unequal variance, and the appropriateness of one­ tailed or two­tailed significance was determined on an individual experiment basis. P­values ≤0.05 were considered significant. Where appropriate, data were expressed as the geometric mean ± SEM, unless otherwise stated. Online supplemental material. Fig. S1 shows dose response of poly(I:C) on the production of NP­specific IgG2c and lack of polyclonal IgG2c pro ­ duction. Data presented in Fig. S2 characterizes the kinetics of MZ B cell depletion by anti­integrin treatment. Online supplemental material is avail ­ able at http://www.jem.org/cgi/content/full/jem.20092695/DC1. The authors wish to thank Paul Waterman, Ginger Woznicki, and Catherine Haluszczak for technical assistance, the NJH flow cytometry facility for cell sorting, and Philippa Marrack and Ross Kedl for reagents. Additionally, we thank Klaus Rajewsky for generosity with reagents and mice. We also appreciate the advice and thoughtful comments from Ross Kedl, John Kappler, and members of the laboratory during these studies. This work was supported by the National Institutes of Health (AI052310 to R. Pelanda and AI052157 and AI078468 to R.M. Torres). The authors have no conflicting financial interests. Submitted: 18 December 2009 Accepted: 17 May 2010 REFERENCES Alexopoulou, L., A.C. Holt, R. Medzhitov, and R.A. Flavell. 2001. Recognition of double­ stranded RNA and activation of NF­ kappaB by Toll­ like receptor 3. Nature. 413:732–738. doi:10.1038/35099560 Amlot, P.L., D. Grennan, and J.H. Humphrey. 1985. Splenic dependence of the antibody response to thymus­ independent (TI­ 2) antigens. Eur. J. Immunol. 15:508–512. doi:10.1002/eji.1830150516 Baccala, R., K. Hoebe, D.H. Kono, B. Beutler, and A.N. Theofilopoulos. 2007. 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