recombinant mouse cd48  (R&D Systems)

Journal: Advanced Science

Article Title: High‐Affinity Superantigen‐Based Trifunctional Immune Cell Engager Synergizes NK and T Cell Activation for Tumor Suppression

doi: 10.1002/advs.202310204

Figure Lengend Snippet: SEB binds to human CD2/CD58 and mouse CD2/CD48 to activate T cells. a) The expression level of hTCRvβ3 in the lentivirus‐transduced Jurkat NFAT‐GFP reporter cells. b) Activation of parental Jurkat NFAT‐GFP reporter cells and Jurkat‐hTCRvβ3 NFAT‐GFP reporter cells treated with anti‐CD3/CD28 antibody or 1 µg ml −1 of recombinant wild‐type SEB for 24 hrs. c) Activation of Jurkat‐hTCRvβ3 NFAT‐GFP reporter cells with 0.3 µg ml −1 recombinant SEB variants for 24 hrs. (n = 3) d) Activation of Jurkat‐hTCRvβ3 NFAT‐GFP reporter cells and other CRISPR knockout cells following treatment with anti‐CD3/CD28 or 1 µg ml −1 of recombinant wild‐type SEB for 24 hrs. (n = 3 to 5) e) Human PBMC proliferation by SEB upon anti‐CD2 blocking. Human PBMCs were pre‐treated with 1 µg ml −1 of an isotype control antibody or anti‐CD2 antibody and then treated with anti‐CD3/CD28 antibody or 1 ng ml −1 of recombinant wild‐type SEB for evaluation. (n = 3) f–i) Protein‐protein interactions between SEB and CD2, human CD58, and mouse CD48 as predicted with AlphaFold2‐Multimer. The ranked 0 and the predicted aligned error (PAE) score for the model ranked 0 are shown. j,k) The binding of wild‐type SEB and SEB variants toward yeast cells displaying CD2, human CD58, or mouse CD48. The mean fluorescence of protein binding on the surface‐displayed population is shown. The error bars represent mean ± SD. Statistical analyses were performed by One‐way ANOVA c) followed by the Fisher's LSD test, d) with Dunnett correction, and (e) unpaired Student's t ‐test. ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For the other immunoreceptors binding affinity tests, induced yeast cells were incubated with 1 µ m human Fc tagged mouse CD28 recombinant protein (SinoBiological, 50103‐M02H), 5 µ m His tagged human CD80 recombinant protein (SinoBiological, 10698‐H08H), 5 µ m His tagged mouse CD80 recombinant protein (SinoBiological, 50446‐M08H), 5 µ m His tagged human CD86 recombinant protein (SinoBiological, 10699‐H08H), 5 µ m His tagged mouse CD86 recombinant protein (SinoBiological, 50068‐M08H), 2 µ m His tagged human HLA‐DRA recombinant protein (Cusabio, CSB‐EP360793HU), 0.5 µ m human Fc tagged human CD2 recombinant protein (SinoBiological, 10982‐H02H), 2 µ m His tagged mouse CD2 recombinant protein (SinoBiological, 50537‐M08H), 0.5 µ m human Fc tagged human CD58 recombinant protein (SinoBiological, 12409‐H02H), and 0.5 µ m human Fc tagged mouse CD48 recombinant protein (SinoBiological, 50415‐M02H) at 4 °C for 2 h. Yeast cells were washed with PBS once and stained with the anti‐human IgG Fc antibody (Abcam, ab131612), the anti‐His tag antibody (Biolegend, 362603), and the anti‐HA tag antibody (Biolegend, 682404) at 4 °C for 30 min.

Techniques: Expressing, Activation Assay, Recombinant, CRISPR, Knock-Out, Blocking Assay, Control, Binding Assay, Fluorescence, Protein Binding

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: Deconvolved confocal fluorescence and interference reflection micrographs (IRM). Samples were stained for macrophage actin (white), CD48 (yellow) and E. coli (red). CD48 exclusively localized within the macrophage membrane including filopodia (see arrowheads and x-z and y-z cross sections). Filopodia (FP) and lamellipodia (LP) – bacteria contacts were suggested by IRM.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Fluorescence, Staining, Membrane, Bacteria

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) Addition of 2% soluble α−D-mannopyranoside inhibitor (αMM) and CD48 antibodies substantially reduced the rate of E. coli phagocytosis. Uptake rates were analysed for 10 independent macrophages during 10 min live cell experiments and normalized by the time-averaged number of bacteria available in the zone explored by filopodia. Mean values are given as horizontal line. (b) Bacterial uptake was observed at all sides of the macrophages and was independent of the direction of fluid flow (0.1 ml/min flow rate/0.06 pN/μm 2 shear stress). (c) Filopodia length distribution as analysed from fixed macrophages. 95% of the filopodia (n = 400) have a length less than 8 μm, which we defined here as the filopodia sensing zone (yellow area).

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Bacteria, Shear

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) Accumulation of type I fimbriated E. coli FimH-j96 bacteria on flow chamber bottom glass surfaces coated with CD48, mono-mannose bovine serum albumin (1 M) and tri-mannose RNaseB (3 M) under varying shear stresses τ (1 pN μm −2 = 1 Pascal or 10 dynes cm −2 ). E. coli accumulation was analysed after 5 min. As negative controls, either 2% of a mono-mannose inhibitor (αMM) was added to the media ( ), or the non-fimbriated E. coli parent strain AAEC191A was used (Δfim, ). (b) Fraction of E. coli FimH-j96 that adhered firmly on CD48, 1 M and 3 M. Bacteria were defined firmly adhering if they moved less than one-half of a bacterial diameter over >30 s. (c) Effect of different FimH variants on bacterial accumulation to 1 M and CD48. While the low binding FimH-f18 strain only adhered to CD48 and 1 M above a critical shear stress, FimH-j96 E. coli accumulated on CD48 without any shear threshold.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Bacteria, Shear, Binding Assay

Journal: Scientific Reports

Article Title: Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism

doi: 10.1038/srep02884

Figure Lengend Snippet: (a) The mannosylated membrane anchored surface receptor CD48 of macrophages specifically binds to the bacterial fimbrial tip adhesin FimH, which contains a single mannose-binding pocket in the lectin domain (Hook). (b) Due to filopodia retractions, the bacterial fimbriae are elongated. As bacteria adhere tightly to substrate surfaces via multiple bonds, the macrophages fail to pull bacteria off the surface via the hook alone. (c) To facilitate uptake, macrophages protrude lamellipodia towards the bacterium to sequentially break the bonds that anchor E. coli to the surface (Shovel). (d) Once the bacterium is completely lifted off the substrate and lies on the lamellipodium, a phagocytic cup is formed to internalize the bacterium.

Article Snippet: 35 mm tissue culture dishes (Corning CellBIND, 3294) were incubated for 75 min at 37°C with either 100 μg/ml mono-mannose-BSA (Dextra Labs, NGP1108), 20 μg/ml RNaseB (Sigma Aldrich, R1153) or 20 μg/ml recombinant mouse CD48 (R&D Systems, 3327-CD) in 0.02 M bicarbonate buffer.

Techniques: Membrane, Binding Assay, Bacteria