mouse anti human cd40 antibody  (ATCC)

Journal: bioRxiv

Article Title: Single-Cell Protein Interactomes by the Proximity Network Assay

doi: 10.1101/2025.06.19.660329

Figure Lengend Snippet: (A) Heatmap of mean Proximity Scores (log2 ratio) of Raji cells showing protein colocalization and self-clustering. Black frames highlight examples of known protein-protein interactions and previously described multi-protein domains. (B) The interaction network of CD19 and CD20, composed of proteins showing mean Proximity Scores above 0.1 with CD19 or CD20. Gray lines indicate mean Proximity Scores above 0.1 between other members of this interaction network. (C) Proximity Scores across Raji cells for the markers CD40, ICAM-1, and CD81. Each point corresponds to the score of a single cell. (D) Representative immunofluorescence (IF) microscopy images of single cells, showing the clustering behavior of CD40, ICAM-1 and CD81. Scale bars: 2 μm. (E) Representative 3D visualizations of PNA cell graphs of single cells showing the distribution of CD40, ICAM-1, and CD81. A darker dot color indicates a high local density of the protein.

Article Snippet: Raji cells were fixed using 1% PFA, washed and analyzed using PNA, or stained for microscopy using either mouse anti-human CD40, mouse anti-human CD81 or mouse anti-human ICAM-1, followed by secondary staining using an anti-mouse AF647-conjugated monovalent nanobody (Chromotek).

Techniques: Protein-Protein interactions, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Single-Cell Protein Interactomes by the Proximity Network Assay

doi: 10.1101/2025.06.19.660329

Figure Lengend Snippet: (A) Workflow for functional characterization of CAR-T cells. CAR-T cells were either kept separate, or were mixed with CD19-positive Raji cells at an 1:1 E:T ratio. Cells were cocultured for either 4h or 24h, after which they were retrieved and fixed using 1 % PFA. Each sample was split into two duplicates and processed with the Proximity Network Assay according to the DP001 CAR Barcoded Antibody spike-in Proxiome kit (v1.00) manual. (B) The CAR-T cell population included both CD4⁺ and CD8⁺ subsets, with ∼40% of cells expressing the CD19 CAR transgene. (C) Proteins displaying positive colocalization (average score ≥0.1) with the CD19 CAR were plotted in a network (the CD19 CAR Proxiome). Positive colocalization scores for protein pairs other than the CD19 CAR have been grayed out for clarity. (D) Left: High-dimensional clustering based on protein abundance data for CAR-T cells alone, 4h cocultured cells, and 24h cocultured cells efficiently separating CD4, CD8 and Raji cells. Right: Cocultured T cells displayed increased levels of ICAM-1. (E) 3D visualization of a single CD8 T cell from the 24h cocultured sample. The cell displays a patchy ICAM-1 distribution where the patch colocalized with B cell markers like CD40, while anti-colocalizing with T cell marker CD8. (F) Illustration showcasing the process of trogocytosis, exchange of plasma membrane between cells, in CAR-T cells. (G) Quantification of tumor patches on the surface of CD8 + non-activated (CD25 - , PD-1 - ), activated (CD25 + , PD-1 - ) and exhausted (CD25 + , PD-1 + ) T cells. (H) An example of a CD8 T: Raji cell conjugate. The T cell (left) displays expression of T cell specific marker CD3e, while the Raji cell (right) expresses B cell marker CD40.

Article Snippet: Raji cells were fixed using 1% PFA, washed and analyzed using PNA, or stained for microscopy using either mouse anti-human CD40, mouse anti-human CD81 or mouse anti-human ICAM-1, followed by secondary staining using an anti-mouse AF647-conjugated monovalent nanobody (Chromotek).

Techniques: Functional Assay, Expressing, Quantitative Proteomics, Marker, Clinical Proteomics, Membrane