Human PBMCs (2 × 105/well) were left untreated or stimulated with CpG plus anti-IgM for 24 hr in the presence Selleckchem R428 of SC-58125 or NS-398. Supernatants were collected and analysed for prostaglandin E2 (PGE2) levels by enzyme immunoassay (Cayman Chemical). Purified human B-cell viability was assessed by 7-aminoactinomycin D (7-AAD) staining using BD Bioscience’s

Cell Viability Solution. Cells were surface stained for allophycocyanin-conjugated CD19 and phycoerythrin-conjugated CD38 (CD38-PE; BD Biosciences, San Jose, CA). Proliferation was assessed by CFSE (Molecular Probes/Invitrogen, Carlsbad, CA) labelling of cells before agonist/drug treatment. Cells were incubated with 5 μm CFSE for 5 min at room temperature and washed three times before stimulation Fulvestrant order in culture for 7 days. For intracellular staining, CD19+ purified human B cells were fixed and permeabilized using the Caltag fix and perm kit (Caltag Laboratories/Invitrogen, Burlingame, CA) and stained for intracellular fluorescein isothiocyanate-conjugated IgM (IgM-FITC) or IgG-FITC (BD Biosciences). Freshly isolated wild-type and Cox-2-deficient mouse splenocytes were stained for CD19-PE (BD Biosciences), CD21-FITC (eBioscience, San Diego, CA) and CD23-biotin (BD Biosciences) to assess marginal zone B-cell populations. Secondary labelling was performed with streptavidin-allophycocyanin (Caltag Laboratories/Invitrogen). Wild-type and Cox-2-deficient B cells were stained

for surface CD138-PE (BD Biosciences) expression after 72 hr of culture. Fluorescently labelled cells were analysed on a FACSCalibur

flow cytometer (BD Biosciences) and results were analysed using FlowJo software (Tree Star Inc., Ashland, OR). Following 24, 48, 72 and 96 hr culture of human B cells (3 × 106 cells/ml), total RNA was isolated using a Qiagen RNAeasy mini kit. RT Superscript III and random primers (Invitrogen, Carlsbad, CA) were used to reverse transcribe isolated RNA to complementary DNA. Steady-state levels of Blimp-1, Xbp-1, Pax5 and 7S (housekeeping control) messenger RNA (mRNA) were assessed by real-time polymerase chain reaction (PCR). Primers used included Blimp-1 sense 5′-GTGTCAGAACGGGATGAAC-3′ and antisense 5′-TGTTAGAACGGTAGAGGTCC-3′, Anacetrapib Xbp-1 sense 5′-TGGCGGTATTGACTCTTCAG-3′ and antisense 5′-ACGAGGTCATCTTCTACAGG-3′, Pax5 sense 5′-TTGCTCATCAAGGTGTCAGG-3′ and antisense 5′-TAGGCACGGTGTCATTGTC-3′ and 7S sense 5′-ACCACCA GGTTGCCTAAGGA-3′ and antisense 5′-CACGGGAGT TTTGACCTGCT-3′. As previously described, iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) was used to quantify amplified products and results were analysed with the Bio-Rad Icycler software.11,12 Blimp-1, Xbp-1 and Pax5 mRNA steady-state levels were normalized to 7S expression. Fold mRNA decrease was determined by comparing mRNA steady-state levels from vehicle-treated peripheral human B cells with SC-58125-treated B cells. Purified normal human B lymphocytes were lysed in ELB buffer: 50 mm HEPES (pH 7.0), 0.