Cd5 pe

Recombinant human BAFF (rhBAFF) was from PeproTech (Rocky Hill, NJ, USA). Rituximab, trastuzumab and bevacizumab were from Roche (Basel, Switzerland), and belimumab was from GlaxoSmithKline (Brentford, UK). In neutralization experiments, binding of belimumab to rhBAFF was allowed for 45–60 min at room temperature prior to adding. Idelalisib and ibrutinib were from Selleckchem (Munich, Germany), and venetoclax was from Active Biochem LTD (Kowloon, Hong Kong). The antibodies CD5-APC, CD5-PE, CD19-FITC and anti-active caspase-3-AlexaFluor647 were purchased from BD Pharmingen (San Diego, CA, USA), CD3-eFluor450 was from eBioscience (San Diego, CA, USA). Rabbit isotype control-AlexaFluor647 was from Cell Signaling Technology (Danvers, MA, USA), LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit for 405 nm was from Invitrogen (Waltham, MA, USA), 7-aminoactinomycin D (7-AAD) and CD3-APCFire^TM 750 were from BioLegend (San Diego, CA, USA). Tetramethylrhodamine ethyl ester perchlorate (TMRE) was purchased from Sigma (St. Louis, MO, USA) and used at a concentration of 50 nM. Primary cells were cultivated in RPMI-1640 GlutaMAX^TM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Biochrom AG, Berlin, Germany) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland).

PBMCs (1 × 10⁶ cells) were incubated with anti-CD19-PEDy647, anti-CD38-PE, anti-CD41a-FITC, anti-CD41a-PE (Immunotools, Friesoythe, Germany), anti-CD19-PECy7, anti-CD27-APC, anti-BAFFR-FITC, anti-TACI-PE (BioLegend, San Diego, USA), anti-CD86-PE, anti-IgG-PE, anti-IgD-FITC (BD), anti-IgA-FITC, and anti-IgM-PE (Dako, CA, USA) mAbs and the corresponding isotype controls. Whole blood (100 μl) was incubated with anti-CD4-PECy7 (BioLegend), CD5-PE (BD Biosciences), CD19-PEDy647, and CD41a-FITC (Immunotools). Red blood cells were lysed, and white cells fixed using BD FACS lysing solution (BD Biosciences) to be analyzed by flow cytometry.

The antibodies used for flow cytometry included: CD4-PE (RM4-5), CD4-v450 (RM4-5), CD5-PE (53-7.3), CD8-APC (53-6.7), CD19-FITC (ID3), CD22-PE (Cy34.1), CD45R/B220-APC (RA3-6B2), CD62L-APC (MEL-14), CD69-FITC(H1.2F3), CD86-PE (GL1), CD93(AA4.1), CD95-PE.Cy7 (Jo2), CD134-Biotin (OX-86), CXCR5-PE.Cy7 (2G8), PD-1-APC (J43), and PNA-FITC (L7281) (all from BD Biosciences, San Jose, CA), IgM-FITC) (R6-60.2 from Southern Biotech), IgD-APC-Cy7 (11-26c.2a from BioLegend, San Diego, CA, USA), CD21/CD35-eFlour450 [eBio4F3 (4E3)], and CD23-PE-Cy7 (B3B4) (both from eBioscience Inc., San Diego, CA, USA). Biotinylated antibodies were detected using FITC-conjugated streptavidin (BD Biosciences). Flow cytometric analysis was performed using various combinations of these antibodies on single cell suspensions of splenocytes. Stained cells were analyzed in the UNMC Flow Cytometry Research Facility using the BD LSR II flow cytometer. Data were analyzed using FACSDiva software, version 8.0.2 (BD Biosciences). For flow cytometry analyses, splenocytes were collected from mice that were 5–6 months of age.

Gum arabic, activated carbon, 2,7-dichlorofluorescein diacetate (DCFDA), toluidine blue, protease inhibitor cocktail tablets, phosphatase inhibitor cocktail tablets, MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel were purchased from Merck KGaA (Darmstadt, Germany). 25% Aqueous Solution Glutaraldehyde, Paraformaldehyde, Sorensen's Phosphate Buffer, 2% Aqueous Solution Osmium Tetroxide, Ethyl Alcohol, Acetone, Araldite, Dibutyl phthalate (DBP) were purchased Electron Microscopy Sciences (Hatfield, USA). Leukocyte Activation Cocktail with BD GolgiPlug™, FACS antibodies include anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, CD45-APC-Cy7, CD3e-FITC, CD4-V450, CD8-BV510, CD25-BV605, IL-4-PE-Cy7, IFN-γ-PE, FoxP3-AF647, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c--AF700 were purchased from BD (Heidelberg, Germany); Another FACS antibody Anti-mouse-IL-17A-BV650 was purchased from eBioscience (Frankfurt am Main, Germany). Anti-mouse HMGB1, Anti-mouse phospho-p65, anti-mouse phospho-p38, anti-mouse GAPDH and anti-rabbit IgG HRP were obtained from Cell signaling Technology (Frankfurt am Main, Germany). IgE and histamine ELISA kits were purchased from Abcam (Berlin, Germany).