GSL expression on the cell surface membranes was analysed by flow cytometry (CyAn ADP Analyzer, Beckman Coulter, Nyon, CH, USA) prior to antibody labelling. Unconjugated antibodies included anti-P1 human IgM (clone P3NIL100; Immucor Gamma, Rödermark, Germany), anti-P1 murine monoclonal IgM (clone OSK17; Immucor Gamma) and anti-Gb3 monoclonal IgG2b (CD77, Pk) (clone BGR23; Seikagaku Biobusiness Corporation, Tokyo, Japan). Biotin-conjugated antibodies included anti-human mouse IgM (BD Bioscience, Basel, Switzerland), rat anti-mouse IgM and rat anti-mouse IgG2b (BD Bioscience). Streptavidin conjugated to FITC (BD Bioscience) was used for fluorescence detection. Dead and apoptotic cells were separated from live cells using propidium iodide (BD Bioscience). Matching isotype monoclonal antibodies conjugated to FITC were used as controls (BD Bioscience). All investigated cell lines were gated individually to exclude debris, followed by single cell gating to remove dead cells and doublets. Data acquisition was performed using Summit v4.3 (CyAn ADP Analyzer, Beckman Coulter). Data analysis was performed using FlowJo v9 (Tree Star Inc., Ashland, OR, USA).
Rat anti mouse igg2b
Manufactured by BD
Sourced in Switzerland
Rat anti-mouse IgG2b is an antibody that specifically binds to the IgG2b subclass of mouse immunoglobulins. It can be used for the detection and quantification of mouse IgG2b in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (1 mentions)
Streptavidin conjugated to fitc, by BD (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Rat anti mouse igg2a, by BD (1 mentions)
Rat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse igg1, by BD (1 mentions)
Streptavidin peroxidase, by BD (1 mentions)
2 protocols using rat anti mouse igg2b
1
Flow Cytometry Analysis of GSL Expression
2
ELISA for E. coli Antibody Evaluation
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For evaluating E. coli -specific antibodies by enzyme-linked immunosorbent assay (ELISA), flat-bottom, high-binding, 96-well enzyme immunoassay (EIA)/radioimmunoassay (RIA) plates (Costar) were coated with nearly confluent, log-phase E. coli UTI89 and allowed to dry overnight under UV light. E. coli -coated plates were blocked with 3% milk and first probed with serum dilutions from each mouse at the indicated dilution and then secondarily probed with the following biotin-conjugated anti-mouse antibodies: rat anti-mouse IgG (eBioscience catalog [cat.] no. 13-4013-8), rat anti-mouse IgM (eBioscience 13-5890-1589), rat anti-mouse IgA (eBioscience 13-5994-82), rat anti-mouse IgG1 (BD Pharmingen cat. no. 553441), rat anti-mouse IgG2a (BD Pharmingen 553388), rat anti-mouse IgG2b (BD Pharmingen 553393), rabbit anti-mouse IgG2c (Invitrogen cat. no. SA5-10235), and rat anti-mouse IgG3 (BD Pharmingen 553401). Each antibody was used at an 1:1,000 dilution and developed with streptavidin-peroxidase (554066; BD Bioscience) using o-phenylenediamine dihydrochloride as a substrate; absorbance at 450 nm (A450) was read as described previously (88 (link)).
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