Following stimulation in a 96-well

flat bottom plate, purified B cells were incubated with 4 μM DHE (Molecular Probes) as previously described by Laniewski and Grayson [45]. Surface staining was performed by incubating the cells in a 1:100 dilution of rat anti-mouse B220-allophycocyanin (BD Pharmingen) in 2% FACS Buffer (phosphate buffered saline plus 2% FCS) for 30 min on ice. Cells were washed three times and fixed in 2% paraformaldehyde (Sigma-Aldrich). Purified (1.5 × 106) selleck kinase inhibitor B cells were seeded into wells containing an air-dried, poly-L-lysine (0.01% solution, Sigma-Aldrich)-coated coverslip for 30 min at room temperature. After washing with PBS, cells were stimulated in the presence or absence of 10 μg/mL anti-IgM or 0.2

mM hydrogen peroxide at 37°C. Additionally, one sample was pretreated with 20 mM NAC for 1 h prior to stimulation. At the end of each timepoint, samples were washed, incubated in vehicle or dimedone, and processed for confocal microscopy according to Seo and Carroll [25] using a 1:500 dilution of anti-dimedone antibody (Millipore) and a secondary goat anti-rabbit Alexa-Fluor 488 (Invitrogen). Following sulfenic acid staining, cells were stained with DRAQ5 (Cell Signaling) and mounted with selleck chemicals ProLong Gold anti-fade reagent (Invitrogen) according to manufacturer’s protocol. 12-Bit images were acquired using a Zeiss LSM 510 confocal laser scanning microscope with a 63× magnification objective lens. For each experiment, exposure settings were determined to avoid saturation and were used for all samples in order to compare intensities. Bay 11-7085 The open source software ImageJ (National Institutes of Health) was used to quantify cysteine sulfenic acid levels within the nucleus and cytoplasm. The mean fluorescent intensity within the borders of the cell and nucleus was determined. To determine the cytoplasmic fluorescence, the nuclear value was subtracted from the whole cell value. Six fields of view were analyzed for each condition. Purified (2 × 106) B cells were stimulated with 10 μg/mL anti-IgM, washed one time with PBS, and lysed in the presence

of 50 mM Tris-HCl, 100 mM NaCl, 20 mM β-glycerophosphate, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal, 0.5% Triton X-100, 1 mM Na3VO4, 20 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM dimedone. After incubating on ice for 30 min, samples were stored at −80°C. For biotin-based affinity capture experiments, purified B cells (4 × 106) were stimulated with 10 μg/mL anti-IgM, washed one time with PBS, and lysed in the presence of 50 mM Tris-HCl, 100 mM NaCl, 100 μM DTPA, 20 mM β-glycerophosphate, 0.1% SDS, 0.5% sodium deoxycholate, 0.5% Igepal, 0.5% Triton X-100, 1 mM Na3VO4, 20 mM NaF, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 200 units of catalase, 10 mM N-ethyl-maleimide, and 5 mM DCP-Bio1 [46].