Miyazaki M, Rivera RR, Miyazaki K, Lin C, Agata Y, Murre C

Miyazaki M, Rivera RR, Miyazaki K, Lin C, Agata Y, Murre C. A (MPL)-based adjuvant (Sigma) (31). For boost immunizations, 200 g NP-KLH in phosphate-buffered saline (PBS) was administered more than 60 days after priming. Sheep red blood cells (SRBC) (Colorado Serum Co.; 31102) were injected into the peritoneum in a 100-l suspension in PBS (10%). Flow cytometry. Single-cell suspensions of bone marrow, lymph nodes, and spleens were prepared, and red blood cells were lysed, counted, and stained with the following antibodies: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, allophycocyanin (APC)-, APC-Cy7-, Pacific Blue-, Alexa Fluor 700-, Alexa Fluor 780-, peridinin chlorophyll protein (PerCP)-Cy5.5-, PE-Cy7-, or biotin-labeled monoclonal antibodies purchased from BD Pharmingen or eBioscience, including B220 (RA3-6B2), CD19 (1D3), CD38 (90), IgD (11.26), GL7 (GL7), CD95 (Jo-2), CXCR4 (2B11), IgM (R6-60.2), CD86 (GL1), IgG1 (A85-1), CD21 (7G6), CD23 (B3B4), c-kit (ACK2), CD25 (PC61), CD138 (281-2), CD93 (AA4.1), Sca1 (E13-161.7), CD150 (TC15-12F12.2), Flt3 (A2F10), interleukin 7 receptor (IL-7R) (A7R34), Ly6D (49-H4), CD8 (53-6.7), Mac1 (M1/70), Gr1 (RB6-8C5), NK1.1 (PK136), Ter119 (TER119), TCR (H57), TCR (GL3), CD3 (2C11), CD4 (GK1.5), and CD8 (53-6.7). Biotinylated antibodies were labeled with streptavidin-conjugated Qdot-605 (Invitrogen). Clone 2.4 G2 anti-CD16-CD32 (eBioscience) was used to block Fc receptors. Dead cells were removed from sorting and analysis by propidium iodide 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 (PI) staining (Sigma-Aldrich). Data were collected on an LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar). Sorting was performed on a FACSAria (BD). ELISA, ELISpot assay, and BrdU labeling. The numbers of antibody-secreting cells (ASCs) were determined as follows. Cells were cultured overnight at 37C on 96-well MultiScreen-HA filter plates (Millipore) precoated with goat anti-mouse Ig capture antibodies (Southern Biotechnology Associates [SBA]). Spots were visualized with goat anti-mouse IgM or IgG1 antibodies conjugated to horseradish peroxidase (HRP), and color was developed with 3-amino-9-ethyl carbazole (Sigma-Aldrich). Serum immunoglobulins and NP-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA). NP-specific ASCs were recognized by 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 enzyme-linked immunospot (ELISpot) assay as explained previously (32). For detection of cycling cells, mice were exposed to the thymidine analogue bromodeoxyuridine (BrdU) (0.8 mg/ml) in drinking water. DZ and LZ GC B cells were stained with Fas, GL7, CD86, and CXCR4; fixed; and stained with FITC-labeled anti-BrdU (Becton Dickinson) as explained previously (33). B TFIIH cell isolation and cell tradition. B cells from spleens were isolated using a negative-selection protocol as explained previously (Miltenyi Biotec). B cells were cultured in RPMI 1640 medium plus 5% fetal bovine serum (FBS), antibiotics, 2 mM l-glutamine, and -mercaptoethanol (50 M). The B cells were activated in total medium at 1 106 cells/ml in the presence of lipopolysaccharide (LPS) (25 g/ml; Sigma; L2654-1MG) and IL-4 (5 ng/ml; RD Systems; 404-ML-101/CF) as explained previously (12). The cells were cultured at 37C and 5% CO2, harvested at the changing times indicated, washed, and analyzed using circulation cytometry. RNA-seq analysis. Transcriptome sequencing (RNA-seq) data were analyzed with the pipeline tool Omics Pipe using the RNA-seq count-based differential manifestation analysis pipeline (34, 35). Quality control of the uncooked fastq documents was performed using the software tool FastQC (Babraham Bioinformatics). Sequencing reads were aligned to the mouse genome (mm10) using the Celebrity aligner (36). Go through quantification was performed in the exon level using htseq-count with UCSC RefSeq annotation (35). The R BioConductor package DESeq2 was used to calculate size factors to normalize library sizes across replicates and to calculate means and variances based on a negative binomial distribution model to detect differentially indicated genes, based on an modified value of 0.05 (37). Functional enrichment of the differentially indicated genes was performed using the ToppGene Suite and 3-O-(2-Aminoethyl)-25-hydroxyvitamin D3 WebGestalt (38). An connection network of the differentially indicated genes in the B cell receptor signaling KEGG pathway and the 20 most connected neighbors was created using relationships from GeneMANIA. Statistical analysis. values were calculated with the two-tailed.