rat anti tfr cd71  (Bio-Rad)

Journal: bioRxiv

Article Title: Proteomic profiling of isolated immune synapses from primary mouse B cells

doi: 10.1101/2023.02.23.529674

Figure Lengend Snippet: (A) Schematic representation of the IS isolation method. Primary mouse B cells are let to form the IS on magnetic beads and the IS structures are extracted for proteomic analysis. (B) Coating efficiency of the beads with selected antibodies was determined using flow cytometry. Single beads (green), duplets (black) and aggregates (orange) were gated separately (on the left) and the fluorescence intensity of the single beads was analysed (on the right). Red histogram: uncoated beads. Blue histogram: coated beads. (C) Cell-bead conjugate formation using beads coated with anti-BCR, anti-TfR, anti-MHC-II, or anti-TfR + anti-MHC-II antibodies. Primary cells were incubated with coated beads for 15 min and analysed using an inverted brightfield microscopy (on the left). Yellow squares highlight single conjugates. After conjugation, unbound cells were counted to get the percentage of conjugate formation (on the right). In the graph, the average percentage of conjugates [100 – (unbound cells/total cells)*100] per experiment are shown, from 2-15 experiments. Anti-BCR: 71,4 ± 1.7 % ( n = 15). Anti-TfR 44.6 ± 4.0 % ( n = 15). Anti-MHC-II: 28.3 ± 4.4 % ( n = 4). Anti-CD71 + anti-MHC-II: 29.0 ± 3.0 % ( n = 2). Each symbol represents and individual experiment. **** P < 0.0001 (unpaired Student’s t-test).

Article Snippet: For bead preparation, 20 x 10 6 Dynabeads™ M-450 Tosyl-activated (#14013, Thermo Fisher Scientific) were washed two times with Buffer 1 (0.1M sodium phosphate buffer, pH 7.4-8) and resuspended in Buffer 1 containing one of the following ligands: AffiniPure F(ab’) 2 Fragment Goat Anti-Mouse IgM, μ chain specific (#115-006-020, Jackson ImmunoResearch) or rat anti-mouse CD71 (TfR) (#553264, BD Bioscience) (Cunha et al., in press ).

Techniques: Isolation, Magnetic Beads, Flow Cytometry, Fluorescence, Incubation, Microscopy, Conjugation Assay

Journal: Molecular Medicine Reports

Article Title: Itgb3-integrin-deficient mice may not be a sufficient model for patients with Glanzmann thrombasthenia

doi: 10.3892/mmr.2021.12088

Figure Lengend Snippet: Spleen extramedullary hematopoiesis and iron deficiency of Itgb3 −/− mice. (A) Relative ratio of each organ to whole body weight in different genotype mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. (B) Relative ratio of spleen to whole body weight in Itgb3 −/− , Itgb3 +/− , Itgb3 +/+ and CHM mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. Immunohistochemical study of spleen biopsy from Itgb3 +/+ , Itgb3 −/− mice. Anti-CD3 antibody was used to label T lymphocytes, anti-CD19 antibody to label B lymphocytes and anti-CD71 antibody to label erythrocytes. Magnification (C) ×100 and (D) ×600. (E) Concentration of plasma ferritin in Itgb3 −/− mice. (F) OD value of fecal occult blood in Itgb3 −/− mice using ELISA. CHM, chronic hemorrhagic model; H&E, hematoxylin and eosin; Itgb3, integrin β3; OD, optical density; ns, not significant.

Article Snippet: Rabbit anti-CD3 monoclonal antibody (cat. no. MAB4841) was purchased from R&D Systems China Co., Ltd. and rat anti-transferrin R (CD71) monoclonal antibody (8D3; cat. no. NB 100-64979-0.05mg) was purchased from Novus Biologicals, LLC.

Techniques: Immunohistochemical staining, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay