Pe anti igg1

Fasting venous blood samples were collected from individual subjects, and peripheral blood mononuclear cells were isolated by density‐gradient centrifugation using Ficoll‐Paque Plus (Amersham Biosciences, Little Chalfont, UK). Peripheral blood mononuclear cells at 1 × 10⁶/tube were stained in duplicate with APC‐H7‐anti‐CD3, PerCP‐Cy5.5‐anti‐CD19, PE‐Cy7‐anti‐CD27, APC‐anti‐CD38, PE‐anti‐CD138, PE‐CF594‐anti‐CD20, FITC‐anti‐IgD (BD Biosciences, San Jose, CA, USA) in the dark at room temperature for 30 min. After being washed, the cells were fixed and permeabilized using a fixation/permeabilization kit (BD Biosciences), followed by intracellular staining with BV510‐anti‐IgG, and BV421‐anti‐IgM (BD Biosciences). Negative controls were stained with isotype‐matched control antibodies (APC‐H7‐anti‐IgG1, PerCP‐Cy5.5‐anti‐IgG1, PE‐Cy7‐anti‐IgG1, APC‐anti‐IgG1, PE‐anti‐IgG1, PE‐CF594‐anti‐IgG2b, FITC‐anti‐IgG2a, BV421‐anti‐IgG1, BV510‐anti‐IgG1; BD Biosciences). The percentages of different subsets of B cells were characterized on a FACSAria II (Becton Dickinson, San Jose, CA, USA) and at least 50,000 events were analysed by FlowJo software (v5.7.2; TreeStar Inc., Ashland, OR, USA). The number of each type of cells tested was calculated, according to the percentages of this type of cells multiplied lymphocyte counts.

The following antibodies and reagents were used at the indicated concentrations. Allophycocyanin (APC)-conjugated or phycoerythrin (PE)-conjugated anti-mouse CD45R/B220 (RA3-6B2; 2 µg/ml), fluorescein isothiocyanate (FITC)- or PE-conjugated anti-IgM (R6-60.2; 5 µg/ml), APC-anti-CD19 (1D3; 2 µg/ml), FITC-anti-IgD (11-26c.2a; 5 µg/ml), FITC-anti-CD21/CD35 (7G6; 10 µg/ml), biotinylated anti-heat stable antigen (HSA; M1/69; 0.1 µg/ml), FITC-anti-CD45.1 (A20; 2 µg/ml), PE-anti-CD38 (90; 0.7 µg/ml), PE-anti-IgG1 (A85-1; 1 µg/ml), PE-anti-CD138 (281-1; 2 µg/ml), PE-conjugated streptavidin (1 µg/ml) or PerCP-5.5cyanine-conjugated streptavidin (0.5 µg/ml) were from BD Bioscience. biotinylated anti-GL7 (GL7; 2 µg/ml) purchased from eBioscience. Alexa Fluor 647-labeled HEL was prepared with an Alexa Fluor647 antibody-labeling kit (Invitrogen). Before staining with the antibodies, cells were incubated for 10 min on ice with anti-Fcγ III/II receptor (2.4G2; 0.5 µg/ml; BD Bioscience) to block Fc receptors. In some experiments, mice were injected intraperitoneally with 300 µl of BrdU (Invitrogen) and sacrificed 5 h later. Splenocytes were then stained with APC BrdU flow kit (Becton-Dickinson). Flow cytometric analyses were done on FACS Calibur (BD Bioscience).

The polyelectrolyte multilayer films were prepared from low molecular weight cationic chitosan (Chi) and anionic chondroitin sulfate (CS) purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cross-linking chemicals i.e., 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydrosulfosuccinimide (NHS) were purchased from Sigma-Aldrich. The human blood was purchased from a donation center. A plasma protein adsorption assay was performed using Qubit^® Protein Assay kit from ThermoFischer Scientific (Waltham, MA, USA). An impact R test kit was purchased from DiaMed Ltd. (Sofia, Bulgaria). Blood coagulation system activation was determined based on a confocal laser scanning microscopy and a flow cytometry analysis after staining with anti-CD62P FITC, anti-CD45 PE, anti-PAC-1 FITC, anti-CD61-PerCP, anti-CD14-PerCP, and anti-CD61-FITC antibodies as well as isotype controls (anti-IgG1-PE, anti-IgM-FITC, and anti-IgG1-FITC) supplied by BD Bioscience (San Jose, CA, USA). A ZymuphenMP-activity ELISA kit from Hyphen Biomed Eragny (Neuville-sur-Oise, France), was applied in a microparticles concentration analysis.