ELISpot/ELISA analysis. For ELISpot analysis, cells were apportioned to PE, NP3-BSA, or polyclonal anti-Ig–coated Multiscreen 96-well plates (Millipore) with twofold serial dilutions made before incubation for 7–18 h at 37°C. PCs were detected by HRP-conjugated or biotinylated anti–mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgE, or IgA polyclonal antibodies (Sout-h ernBiotech.), with streptavidin-HRP used in a second step when necessary. ELISpots were developed as previously described (Benson et al., 2008). For ELISA analysis, plates were coated with either 10 µg/ml PE or 4 × 160 PFU of H2O2-inactivated VACVWR (Hammarlund et al., 2008) overnight in PBS, blocked with PBS + 5% FBS, and washed, and then 1:2,000 diluted serum added at serial 1:2 dilutions. Serum from either a PE hyperimmunized mouse or from a VACVWR mouse was included on each plate as a reference between plates and between experiments and used to generate a standard curve, with these serum allotted the value 8,000 arbitrary units. Antibody levels were detected with IgG-AP (SouthernBiotech) and developed with 1 mg/ml pNPP (Sigma-Aldrich) in 0.05 sodium carbonate buffer. Immunofluoresence microscopy. Spleens were presoaked in 15% sucrose/PBS for 30 min and snap-frozen in OCT compound (Sakura Finetek) in a dry ice/ethanol slurry and stored at80°C. Tissues were sectioned onto slides using a cryostat with 8-µm-thick sections. After drying at room tem- perature, the tissue was fixed in ice-cold 1:1 acetone/methanol mix for 10 min and allowed to dry, after which sections were outlined with an ImmEdge Pen (Vector Laboratories). Sections were blocked with 10% heat-inactivated normal rat serum/PBS for 1 h, washed, and stained with IgD-Alexa Fluor 647, GL7-FITC, and CD35-biotin, followed by staining with SA-PerCP. After washing, slides were mounted with PermaFluor Aqueous Mounting Media (Thermo Fisher Scientific). Samples were analyzed on a LSM 510 META confocal microscope using LSM software (Carl Zeiss, Inc.). Online supplemental material. Fig. S1 shows FACS gating scheme and phenotype of PE-BMEM. Fig. S2 displays the FACS profiles and ga-t ing schemes used to sort-purify PE-BMEM, NP-BMEM, and NF B cells. Fig. S3 contains FACS profiles showing BrdU incorporation by PE-BMEM and data corresponding to experiments presented in Fig. 2. Fig. S4 con- tains FACS plots showing BrdU incorporation by PE-BMEM and D10 GC B cells, with these data corresponding to experiments presented in Fig. 3. Fig. S5 contains FACS plots showing BrdU incorporation by PE-BMEM and NF B cells after in vivo administration of TLR agonists, CD40 agonists, and bystander T cell help. Data correspond to experiments presented in Fig. 4. Fig. S6 depicts the presence of VACVWR-specific CD4 and CD8 cells and VACVWR–specific serum IgG in infected hosts. FACS plots are also depicted showing levels of BrdU incorporation by PE-BMEM after protein vaccination or viral infection, with data corresponding to experiments de- picted in Fig. 5. Online supplemental material is available at http://www.jem .org/cgi/content/full/jem.20090667/DC1. We thank Dr. Klaus Rajewsky and Dr. Stefano Casola for providing the C1-cre mice. We thank Dr. Stacey Dillon and ZymoGenetics, Inc. (Seattle, WA) for providing some of the reagents used in these studies. This work was supported by National Institutes of Health (NIH) grants RO1A1026296 and RO1A108896 to R.J. Noelle and NIH pre-doctoral training grant T32AI07363 to M.J. Benson. 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